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Authors: Ilse Pretorius
Group: Electronic Pathogen Detection, XPRIZE Rapid Covid Testing
Summary: Electronic pathogen detection (EPD) is a non - invasive, rapid, affordable, point- of- care test, for Covid 19 resulting from infection with SARS-CoV-2 virus.EPD scanning technology, is a completely novel technique, designed to assess the human body for the presence or absence of a pathogen.The technology implements a unique scanning method, by delivering an extremely low electrical current to the skin, and then recording the resulting physiological response to this stimulation, from the skin.Although the technique is unique, there are similarities to existing technologies that can be used to explain the modus operandi, of this novel technology. The microcurrent delivery component of the diagnostic technology can be loosely described as containing elements analogous to Microcurrent Electrical Therapy (MET) and TENS (Trans dermal electrical nervous stimulation), whereas the response component of the technology can be seen as similar to Electrocardiography (ECG), Elelctro-enchephalogram (EEG) the Electrodermal Response (EDR), and Body Impedance Assessment (BIA).All of these modalities are widely used, considered safe and have been extensively researched.The skin offers convenient access to study the nervous system, not only is it richly innervated, but it also originates from the same embryonic layer as the nervous system. It is well known that the immune system and immune response is regulated by the neurological system.EPD technology uses this physiological platform offered by the skin to access real-time information of the immune response and compares this physiological information to electronically generated pathogen molecular patterns via a proprietary algorythm.The resulting dermal response , is recorded and displayed on a computer screen as a graph.The graph consists of peaks and troughs.This graph is then statistically verified and analyzed in a proprietary manner.The result of this analysis is then compared to the pathogen dataset that is pre-loaded in the software.This pathogen datatset is derived from the pathogen genes, and a proprietary method, so that the ectodermal response can be compared to the pathogen genes.Once the comparison is complete, the result is displayed.EPD scanning can be performed on humans and animals with consistent results.EPD scans have been performed on adults (all ages) and babies, toddlers and children.EPD scans have been performed on pregnant animals with no adverse results or outcomes to the fetus and newborn.Full Protocol and background:Pathogen Diagnosis The current SARS-COV-2 world pandemic highlights the need for widespread, fast and accurate, point of care testing, and diagnosis. Health experts across the globe agree, that the information gained from testing, is crucial in the management of the epidemiological curve, curbing the spread of the infection, reducing mortality, and optimal use and implementation of medical resources and staff.It is imperative to identify individuals with current infections, testing both symptomatic and asymptomatic individuals, to stop the human to human transmission, of the virus. To date, no country in the world has had the luxury of implementing blanket testing. Mainly because of the steep inclination of the epidemiological curve, whereby governmental and healthcare systems, cannot keep up with the rapid spread of the virus resulting in the exponential increase in symptomatic individuals requiring hospitalization and intensive care. Further challenges include logistical issues, in the distribution of testing kits to existing laboratories, setting up new facilities for sampling and testing, as demand increases. Lastly, third world countries have the added constraints of under-resourced healthcare systems and financial limitations to purchase enough molecular testing equipment.Even if blanket testing were possible, diagnosing the SARS-COV-2 infected individual accurately remains a challenge.This challenge exists for the following reasons: properties unique to the SARS-COV-2 virus (incubation period, a large portion of asymptomatic infected individuals spreading virus), the sensitivity of the RT-PCR ASSAY laboratory tests (depend on which combination of target genes are included in the various test kits, viral load present at the time of sampling, and availability of kits).A positive RT-PCR ASSAY test confirms the presence of SARS-CoV-2 viral nucleic acids (RNA), in a nasopharyngeal swab, oropharyngeal swab, or any bodily fluid or tissue. Generally the sensitivity (accuracy) of RT-PCR ASSAY for SARS-COV-2 is in the region of 80%, with high specificity. Therefore individuals with symptoms of SARS-COV-2 who test negative (up to 20%), should not be managed on the test result alone, but also taking into account epidemiological risk factors (travel history, contacts), clinical symptoms, and by retesting the patient using a different sampling site.Demonstrating seroconversion, the presence in serum of either IgM or both IgM and IgG or IgG, for SARS-COV-2, indicates a past (albeit recent past) exposure, viremia, and production of antibodies. Whether these antibodies are neutralizing and confer immunity is still being established. This type of test cannot be used to identify active infection.Due to costs and the massive logistical demands on RT-PCR ASSAY producing companies (collection kits, purification units, extraction units, reagent solution production), RT-PCR ASSAY is only used in a certain portion of the population at this stage.NICD guidelines for RT-PCR ASSAY testing published on their website https://www.nicd.ac.za/wp-content/uploads/2020/04/COVID-19-Quick-reference-v12-09.04.2020_final-1.pdf:Criteria for the person under investigation (PUI), i.e. a person to be tested for COVID-19 Persons with acute respiratory illness with sudden onset of at least one of the following: cough, sore throat, shortness of breath or fever [≥ 38°C (measured) or history of fever (subjective)] irrespective of admission status.There is an international upsurge and call for emergency research protocols being submitted and approved for clinical trials of new drugs, vaccine development studies and other technology to combat the spread and loss of life caused by this pandemic. EPD is Novel technology for pathogen diagnosisEPD scanning technology, is a completely novel technique, designed to assess the human body for the presence or absence of a pathogen.The technology implements a unique scanning method, by delivering an extremely low electrical current to the skin, and then recording the resulting physiological response to this stimulation, from the skin.Although the technique is unique, there are similarities to existing technologies that can be used to explain the modus operandi, of this novel technology. The microcurrent delivery component of the diagnostic technology can be loosely described as containing elements analogous to Microcurrent Electrical Therapy (MET) and TENS (Trans dermal electrical nervous stimulation), whereas the response component of the technology can be seen as similar to Electrocardiography (ECG), Elelctro-enchephalogram (EEG) the Electrodermal Response (EDR), and Body Impedance Assessment (BIA).All of these modalities are widely used, considered safe and have been extensively researched.The skin offers convenient access to study the nervous system, not only is it richly innervated, but it also originates from the same embryonic layer as the nervous system. It is well known that the immune system and immune response is regulated by the neurological system.EPD technology uses this physiological platform offered by the skin to access real-time information of the immune response and compares this physiological information to electronically generated pathogen molecular patterns via a proprietary algorythm.Background:Immune systemThe immune system consists of cells and proteins found in the blood, lymphatic system and tissue. These cells originate from the bone marrow, and mature in the thymus and spleen.The purpose of the vertebrate immune system is protection from invading pathogens. This protection relies on the innate immune system's ability to co-ordinate effective cell surveillance and recognition of pathogens. The ultimate goal is the eradication of the pathogen, and the production of a neutralizing antibody via the adaptive immune response, this will eventually lead to protection against disease from the same pathogen, if encountered a second time. (Zinkernagel 2002), (Chaplin 2006).Embryology of the immunes systemThe three components of the immune system originate independently of each other from different parts of the mesoderm. macrophage mesoderm, lymphatic, and hematopoietic systems splanchnic mesoderm. This co-ordination depends on chemical signalling between cells, and then recognition of pathogens by cells.Ralph Steinman, Bruce Beutler and Jules Hoffman, were awarded the 2011 Nobel Prize for Physiology in Medicine, for their contributions towards better understanding the interactions between the innate and adaptive parts of the immune response. In particular Ralph Steinman by uncovering that dendritic cells are the bridging communication link, between the cells of the innate and adaptive immune systems.Jules Hoffman and Bruce Beutler for their work on demonstrating how the innate immune system is activated. The innate immune system recognizes conserved Pathogen Associated Molecular Patterns (PAMP's) through Toll-like receptors (TLR's) expressed on the cell surfaces of immune cells. The concept of PAMP's was first coined by the immunologist CA Janeway in 1989, at a Cold Spring Harbor Symposium on Quantitative Biology, (Beutler 2011),( (Paul 2011). Steinman 2012),(Hoffman 2011) (Lemaitre 1996),(Murphy 2017). Cell communicationCells communicate in various ways. Apart from the chemical and molecular, receptor-ligand model of cellular communication, that forms the backbone of our current understanding of biochemical and immunological processes in modern science, there is also a large and rapidly growing body of evidence, that there are additional and equally effective forms of cellular communication, in biological systems.Alexander Gurwitsch was the first scientist to show that onion plant roots communicate with “mitogenic radiation”, radiation in the ultraviolet light range of the electromagnetic frequency spectrum.(Gurwisch, 1923),(Gurwisch, 1926).Professor Fritz Alexander Popp continued this line of investigation and contributed a large body of work on biophotons (photons of light in the ultraviolet and low visible light range, produced by biological systems).(Popp et al., 1994.),(Popp 1988.),(Popp and Klimek 2007).Following on Popp’s work, biophotons and cell communication have been extensively researched by other authors, and in their online book Fields of the Cell (Fels et al., 2015), their work is discussed in great detail.Irene Cosic describes in her book "The resonant recognition model of macromolecular bioactivity: theory and applications" – an alternative resonant model for cell communication, a model for digital biological activity including, it's potential applications, (Cosic 1997) this model, additional to the current model, of ligands and receptors, describing biological processes.Robert Becker the American orthopaedic surgeon, answered the question of how a lizard perfectly grows back an amputated limb. By implementing meticulous research procedures, he showed that following trauma, tissue electrical fields produced at the site of injury, provides an information and communication platform, for cells, to regenerate tissue and amputated limbs to their original specifications. (Becker and Snelden 1998).In his paper “The Electrical Embryo”, Colin Lowry discusses that electrical fields are essential in embryonic growth, pattern and shape (Lowry 1999).Cells react to electrical stimulation, fields and frequenciesIt has also been shown that electrical stimulation, affects cell membrane receptors and ATP production. (Seegers at al. 2001) demonstrated that a microcurrent DC electrical field of less than 150Hz, interacts with HeLa cell receptors, producing a 50-fold increase in ATP in vitro, and a 163% increase in vivo in plasma.Similar findings of electric frequencies activating receptors were published by (Wolf-Goldberg et al. 2013.) showing that Epidermal Growth Factor Receptor (EGFR) activation can be induced by applying a short train of pulsed low strength electric field (LEF) frequencies with 10v/cm pulse-width for 180µs, at 500Hz, for 2min.The idea of using electronic devices, including biosensors to detect pathogenic bacteria is not new and this concept was discussed comprehensively in a review article by (Ivnitski et al.1999). Technological advances since then, including nanotechnology, has opened new doors and new diagnostic possibilities.(Patolski et al. 2004.) using nanowire field resistance, demonstrated that sensitive detection of a single virus is possible by coating a nanowire with Influenza specific antibodies and then adding an Influenza virus (antigen) to this coated nanowire, conductance was observed and a flow of current was demonstrated upon molecular docking, with the specific antibody binding to the virus.Similarly using a slightly different technique, namely impedance spectroscopy, (Abdelghani 2011) demonstrated sensitive detection of the Rabies Virus.The Nervous SystemConsists of the Central end Peripheral nervous system. The central nervous system – the brain and spine, and the peripheral nervous system consists of the somatic and autonomic nervous system. The autonomic nervous system (ANS) is divided in the Sympathetic (SNS) and Parasympathetic nervous system (PNS). Embryologically the nervous system develops from the ectoderm. (Sadler 2011).Immunity is regulated by the Neurological systemThe nervous and immune systems are in constant two-way communication.Our current knowledge and understanding of the immune response, and the regulator thereof, through neuro immune interaction, is based on a large body of scientific work done over the last decade.The primary function of the autonomic nervous system (ANS) is to help the body adapt to internal and external environmental stimuli and demands, by maintaining a healthy balance. (Beissner et al. 2013).The sympathetic nervous system stimulates increased metabolic output.This is known as sympathetic arousal and is characterized by elevated heart rate, blood pressure, sweating and, shunting of blood from the internal gastrointestinal reservoir to the heart, brain and, skeletal muscle, in preparation of a "fight or flight" response.The parasympathetic nervous system effects the opposite, by inducing conservation of metabolic energy and restoration.The brain and immune system are in constant two-way communication to maintain health, and the sympathetic nervous system plays an integrative role in the regulation of the immune response. This physiological equilibrium between the two systems is regulated by two pathways.Centrally, via the Hypothalamic Pituitary Axis Adrenal Corticotropin Pathway (HPA-CRH) and peripherally via the autonomic nervous system (ANS), which in turn is comprised of the sympathetic (SNS) and parasympathetic (PNS) nervous systems. (Dantzer 2019).Loeper and Crouzon demonstrated in 1904 that subcutaneous injection of epinephrine (adrenaline) causes a marked leucocytosis in humans.Primary and secondary lymphoid organs receive substantial sympathetic innervation.With sympathetic nervous system activation, neurotransmitters are released from sympathetic nerve endings in the lymphoid organs, they then bind to specific receptors on lymphatic cells, not only affecting cell traffic, division and, circulation, but also the production of cytokines. This may modulate the type of immune response to either a Th1 cell or Th2 cell type. (Pavlov et al. 2003), (Kin and Sanders 2006).Studying the sympathetic nervous system and particularly the sympathetic response has been ongoing since the 1970's using a variety of biofeedback systems.The most widely studied and used of these methods must be the Galvanic Skin Response, otherwise known as Skin Conductance or Electro Dermal Activity (EDA), (Dawson 2007).Furthermore, T-lymphocytes have also been found to have B2 adrenergic receptors), indicating a neurological regulation of the immune response. (Fan and Wang 2009).The Integumetary systemConsists of the skin and its appendages – hair and nails. The purpose of this system is to protect the body from various kinds of damage, such as loss of water or external harm. Apart from these it also has other functions; thermoregulation, waterproofing, protecting deeper tissues, and waste excretion. The skin is richly innervated with sensory receptors for pain, temperature, pressure and sensation. Embryologically the skin originates from the ectoderm. (Langman’s Essential Medical Embryology, 2011).The skin is the largest organ in the human body, the first defence barrier between the human body and the external environment offering protection against pathogens, chemicals, toxins, and is also richly innervated with about 2500 per cm² nerve endings in the fingertips. (McBride and Schmorrow 2005).Lastly, the integumentary system synthesizes vitamin D.The skin utilizes solar energy which is transmitted as electromagnetic waves. These waves are classified according to their wavelength and frequency. The skin makes use of these properties of the electromagnetic spectrum to produce Vitamin D. (Cavalcanti Soriano Coutinho et al. 2019)Microcurrent electrical therapy (MET)Microcurrent is used for wound healing and pain relief. (Wirsing et al.2013)Application of subsensory micro-amperage current (300 to 500 micro amps pulsed at 0.1 to 680 Hz) to tissue, increases cellular output of ATP. (Seegers et al. 2002)These currents compliment and augment the physiological tissue electrical fields produced at the site of injury which provides an information and communication platform, for cells, to regenerate tissue and amputated limbs to their original specifications. (Becker and Snelden 1998).TensThis technology is widely used for pain management.Application of milliamp current, to blocks pain messages, relayed by sensory nerve fibres, A beta (Aβ), (afferent: to the brain), thereby reducing the ‘c’ fibre (efferent: from the brain) noxious stimulus transmission (pain sensation). The two primary pain relief mechanisms which are targeted are: the “Pain Gate Mechanism” and the “Endogenous Opioid System”. Endorphins (endogenous opoids) are produced in the brain, upon peripheral nervous stimulation.The current intensity (A) (strength) will typically be in the range of 0 - 80 mA up to 100mA. The current is pulsed (the pulse rate or frequency (B) will normally be variable from about2 – 150 Hz. (Vance et al. 2014.)Electrocardiogram (ECG)The heart is a self-powered pump, cardiac pacemaker cells, produce a pace-making current (action potentials) of up to about -50 millivolts, with an amplitude of about 5 nano amperes. This current travels from pacemaker cells via conducting fibers, through the heart muscles, triggering muscle contractions (pumping actions). This current can be measured from the skin with electrodes, (the electrolyte rich fluid of the body, is a good conductor of current). This current of about 1 millivolt, is displayed as a graph the ECG – and represents the sum of all the electrical potentials generated by pacemaker cells at any given time. (Fye 1994)Electroenchephalogram (EEG)Neurons communicate electrically and chemically. Mammalian neurons, produce action potentials of up to about -55 millivolts, with an amplitude of about 1 nano ampere. This can be seen as units of power or watts, or 1.05 x 10^-10 W / neuron. There are billions of neurons in a human brain. Total brain wattage can be calculated as, (1.05 x 10^-10 W / neuron) x (800 million neurons / brain) = 0.085 Watts / brain. The electrical activity in the brain can be recorded via scalp electrodes and displayed on a graph like an ECG, where voltage is plotted against time. The general voltage range of the scalp EEG lie between 10 and 100 µV.(Nunez 2006). (Bronzino, 1999).Electro Dermal Activity (EDA)Electrodermal activity (EDA) is the umbrella term used for defining autonomic changes in the electrical properties of the skin. Historically the two principles used in modern-day skin conductance were discovered by Féré in 1888, who demonstrated decreases in skin resistance with different sensory stimuli, and the Russian physiologist Tarchanoff in 1890, who showed changes in electrical conduction between two skin electrodes during sensory stimulation.(Fere 1888),(Dawson 2007).Skin Conductance or EDA has been shown to provide a sensitive index of the status of the sympathetic nervous system. (Critchley et al., 2000.), (Critchley, 2002).Sweat glands or eccrine glands (sudomotor) are innervated by sympathetic post-ganglionic fibers, consisting of non- myelinated class C fibers. Activation of these nerve fibres modulates sweat secretion. (Figure 1). EDA "taps" into these nerve fibres, and therefore into the sympathetic nervous systemCorrelation between EDA and functional MRI has shown an extensive network of the SNS. (Beissner et al., 2013.), (Critchley et al., 2002). A recent study at Harvard University's Massachusetts Institute of Technology (MIT) by Professor Rozalind Pickard, whilst investigating emotional responses in Autistic Children, revealed an unexpected finding, that EDA can predict the onset of Sudden Death Epilepsy (SUDEP) 24 hours prior the event. SUDEP is a life-threatening condition with cardiac arrest, following post-ictal generalized electrical suppression (Sarkis et al., 2013).Following this discovery, the EMBRACE bracelet warning system was developed. (Poh, 2011.),(www.empatica.com).Bioelectrical Impedance Analysis (BIA) and EPD scan measuring techniqueBioelectrical Impedance Analysis (BIA) takes advantage of the conductive properties of the body, and is a method extensively used in studies assessing body composition, water, fat, muscle. (Kyle 2004),(Mialich et al. 2014).The body’s response to electric current, is similar to an RC circuit, where body fluids (intracellular water (ICW) and extracellular water (ECW)) are represented by resistors and cell walls by capacitors. The impedance measurement is generally measured from the wrist to the contralateral ankle and uses either two or four electrodes. A small current on the order of 1-10 μA is passed between two electrodes, and the voltage is measured between the same (for a two electrode configuration) or between the other two electrodesFat cells have very high impedance or resistance compared to muscle cells, organ cells, extracellular components (including connective tissue). Therefore, electric current will mostly propagate through the fat-free body mass and not the fatty tissue. This means that BIA measures the volume of fat-free mass.EPD technology a combines existing and novel technology for pathogen diagnosis.EPD technology is utilizes the principles of electric stimulation and the resulting response thereto, to investigate the real-time immune response to pathogens.However EPD technology goes further in that it can identify the presence or absence of a pathogen in the mammalian body from these readings.Frequencies are applied in the range of 10 kHz to 500 kHz in a proprietary manner. The body’s reaction is measured and statistically analyzed using propriatory algorythms.This measuring technique uses voltages in the range of 6-volt peak to peak (similar to a line-up of 4 penlight batteries) generating a body current of approximately 500 micro-amp (500 millionth of one amp), at applied frequencies. This well below all safety standards, and is proven to be safe for human subjects. (ICNIRP,1998).Molecular identification of PathogensWatson and Crick received the Nobel prize in 1962 for "for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material." Kerry Mullis in recognition of his invention of the polymerase chain reaction technique, shared the Nobel Prize in 1993 in Chemistry with Michael Smith. (Crick 1962),(Mullis 1993),(Watson 1962).These two scientific milestones paved the way for modern day molecular biology and medicine and our understanding of diseases.It is now possible to sequence the genomes of pathogens and their proteins rapidly, and this information is uploaded to large open domain databases.EPD technology makes use of this information.For the purpose of the Xprise competition, the complete sequenced genome, all genes of SARS-CoV-2 virus will be implemented. However due to the unique properties of EPD technology, it is possible to target not only the viral genome, but also the proteome, and immune response including, lymphocyte antigens, cytokine production, to the viral infection. Cytokines identification, and individual proteins testing are scarce resources and not widely available for data validation in South Africa. EPD technology makes use of an AI model taking all of the above into account.This approach has been used in a Pilot Study using EPD technology on Bovines infected with Mycobacterium Bovis.(Michel et al.2018)SafetyThe safety of bioelectrical instrumentation is assessed by two parameters. One is the aspect of electrical isolation from ground potentials for the subject. The second is the definition of what is a harmless current vs. frequency that can be deliberately introduced into the subject. There are few references that have explicitly established the standards for what is a safe subject current and frequency. (Geddes andBaker, 1975) in Applied Biomedical Instrumentation, describe the threshold of electrical perception of alternating currents of varying frequency.There have been many applications of electrical impedanceat frequencies from 10 KHz to 5 MHz that have been introduced to critical human organs. (Nyboer and Kornmesser, 1970) applied an impedance plethysmograph (designed and built by Rudolph J. Liedtke) to the area of the uterus, to monitor pregnancy labor movements. There were no reported abnormalities after these observations using this method. The instruments that were used had a frequency of 100 KHz (crystal controlled) at approximately 3 milliamps. (Bishop and Nyboer,1970) applied the same instrument directly to the eye with an electrode array, configured in a contact lens, with no ill effects at this frequency and current (100 KHz at 3 milliamps).The EPD frequency in the range of 10 kHz to 600 kHz. This measured body reaction, is mathematically analyzed to extract information, that seems to bear a relationship between certain frequencies, and viruses, that have activated the body’s immune system at the time of measurement.This measuring technique, uses voltages in the range of 6 volt peak to peak (similar to a line-up of 4 penlight batteries) generating a body current of approximately 500 micro-amp (500 millionth of one amp), at applied frequencies.This well below all safety standards, and is proven to be safe for human subjects.(ICNIRP,1998).Body exposure to applied electrical signals: EPD Tracer·Electrical Current used for measurement : 100 to 800 micro-amp induced by applying up to 6 volts·Applied Frequency: 10 to 600 Kilo Hertz·Emf radiation : None – direct application of measuring currentEDR Electro dermal activityElectrical Current used for measurement : NoneSensor Frequency: 4 HertzEmf radiation : None (BIA) Body Impedance Assessment·Electrical Current used for measurement : 100 to 800 micro-amp using up to 6 volts·Applied Frequency: 2100 to 600 Kilo Hertz·Emf radiation : None – direct application of measuring currentTENS Trans dermal electrical nervous stimulation·Electrical Current used for measurement : up to 50 milli-amp using up to 9 volts·Applied Frequency: 250 Hertz pulses with pulse duration .01 to1 milli -seconds·Emf radiation : None – direct application of measuring current.EPD Modus Opperandi:Introduction Gene TargetsAll sequenced genes of the SARS-CoV-2 virus.Note: Make sure the SARS-CoV-2 virus text file is correctly uploaded into the EPD software.Equipment-EPD scanner-Transmitting electrode-Receiving electrode-Laptop with EPD software-Patient-Table-Chair x 2-PPE-Alcohol based surface disinfectant-Hand sanitizerEPD software-EPD 101ProcedureStep 1:Switch on EPD scan unit and PC, connect EPD scan unit to PC.Note: Make sure both PC and EPD scanner are on, and connected.Step 2:Open EPD software.Note: Make sure both PC and EPD scanner are on, and connected.Step 3:Enter patient details into EPD software.Note: Step 4:Connect receiving electrode to LT middle finger terminal phalange of patient. Give patient transmitting electrode to hold in RT hand.Note: Clean electrodes with alcohol based disinfectant. Make sure hands are clean and skin is dry.Step 5:Commence scan.Note: Make sure the EPD software reads the EPD scan machine.Step 6:Read the result.Note:Step 7:Communicate the result.Note: TroubleshootingIf problems getting scan to initiate:Check that scanner is on and connected. Make sure skin is clean and dry.Make sure probes are clean and dry.Time TakenFrom the time that the scan is commenced, to final result takes around 8 minutes. Anticipated ResultsAccurate diagnosis of the presence or absence of SARS-CoV-2 virus.References:ABDELGHANI, A.,2011.Electrochemical Biosensors for Virus Detection, Biosensors for Health, Environment and Biosecurity, ed. 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Sat. 5 Sep 2020. https://www.nobelprize.org/prizes/chemistry/1993/mullis/lecture/MURPHY.K, WEAVER, C, JANEWAY.C, 2017Janeway's Immunobiology9th editionGarland Science New York NYBOER, J., KORNMESSER, G.D., 1970.Electrical Impedance of the abdomen during Maternal LaborAnnals of the New York Academy of Sciences International Conference on Bioelectric Impedance, 170, Pages 801–803.PAUL.W.E.,2011.Bridging Innate and Adaptive ImmunityCell, 147,(6),1212-1215.PATOLSKY, F., ZHENG, G., HAYDEN, O., LAKADAMYALL, M., ZHUANG, X., LIEBER, C.M., 2004.Electrical detection of single virusesPNAS ,101,(39),14017-14020.PAVLOV, V.A., WANG, H., CZURA, C.J, FRIEDMAN, S.G., TRACEY, K.J., 2003.The Cholinergic Anti Inflammatory Pathway: A Missing Link in Neuro-immunomodulationMolecular Medicine, 2003,(9), 5-8.POH, M-Z.,2011.Continuous Assessment of Epileptic Seizures with Wrist-worn Biosensors(Doctoral Thesis)Retrieved from: https://dspace.mit.edu/handle/1721.1/68456POPP, F. A., 1988. Biophoton emissionExperientia ,Basel, 44, 543–544. POPP, F. A., CHANG, J.-J., GU, Q., Ho, M.-W.,Nonsubstantial biocommunication in terms of Dicke's theory BIOELECTRODYNAMICS AND BIOCOMMUNICATIONHo, M.-W., Popp, F. A., Warnke, U., ed, 1994:World Scientific, Singapore. POPP, F. A. & KLIMEK, W., Photon Sucking as an Essential Principle of Biological Regulation BIOPHOTONICS AND CHOERENT SYSTEMS IN BIOLOGY Beloussov, L. V., Voeikov, V. L., Martynyuk, V. S, ed,2007: Springer, New York POPP, F. A. & Yan, Y., 2002. Delayed luminescence of biological systems in terms of coherent states. Physics Letters, A, 293, 93–97. SADLER,T.W.LANGMAN’S ESSENTIAL MEDICAL EMBRIOLOGY201112th Edition Lippincot Williams and Wilkins SARKIS, R., THOME-SOUZA, S., POH, M., KLEHM, J., VANNAN, D., MADSEN, J., PICKARD, R., LODDENKEMPER,T., REINSBERGER,T., 2013.Autonomic changes following generalised tonic clonic seizure: and analysis of adult and paediatric patients with epilepsy. American Epilepsy Society Annual General Meeting, Washington DC, Abstract 2.069SCHOLKMANN,F., FELS,D., CHIFRA,D., 2013.Non-chemical and non-contact cell to cell communication: a short reviewAmerican Journal of Translational Research 5,(6),586-593.SEEGERS, J.C., LOTTERING, M.L., JOUBERT, A.M., JOUBERT,F., KOORTS, A., ENGELBRECHT, C.A., VAN PAPENDORP, D.H., 2002.A pulsed DC electric field affects P2-purinergic receptor functions by altering the ATP levels in in vitro and in vivo systemsMedical Hypotheses, 58,(2),171-176.STEINMAN,R., 2011Nobel Lecture. NobelPrize.org. Nobel Media AB 2020. Sun. 6 Sep 2020. https://www.nobelprize.org/prizes/medicine/2011/steinman/lectureSTEINMANN.R. 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Proper citation: Ilse Pretorius 2020. EPD Electronic Pathogen Detection. protocols.io https://dx.doi.org/10.17504/protocols.io.bkgzktx6 Copy   

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Authors: Ronaldo De Carvalho Augusto, Aki MINODA, Christoph Grunau
Summary: We describe here a step-by-step protocol for the generation of sequence-ready libraries for the Assay for Transposase Accessible Chromatin (ATAC-seq) that provides a positive display of accessible, presumably euchromatic regions. The protocol is straightforward and can be used with small individuals such as daphnia and schistosome worms, and probably many other biological samples of comparable size. This makes the method suitable for population epigenomics studies. This protocol is based on the protocol from Corces et al. (2016), Buenrostro et al. (2015) and Nextera DNA Library Prep Kit (2017) with some modifications. Controls must be done without organisms as input.

Proper citation: Ronaldo De Carvalho Augusto, Aki MINODA, Christoph Grunau 2021. A simple ATAC-seq protocol for population epigenetics. protocols.io https://dx.doi.org/10.17504/protocols.io.bae6ibhe Copy   

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Authors: Chuan-Tsai Tsai, Ya-Wen Lu, Ruey-Hsing Chou, Yi-Lin Tsai, Ming-Ren Kuo, Jiun-Yu Guo, Chi-Ting Lu, Chin-Sung Kuo, Po-Hsun Huang
Summary: Abstract .justify:after { content: ""; display:inline-block; width: 100%; } Introduction: To evaluate efficacy of antithrombotic agents in critically ill patients with elevated troponin I level during intensive care unit (ICU) admission. .justify:after { content: ""; display:inline-block; width: 100%; } Methods and results: It was a retrospective observational study which was conducted in a tertiary teaching hospital in Taipei, Taiwan. All patients hospitalized in ICU for >3 days and with available serum troponin I data from December 2015 to July 2017 were included. Patients with definite diagnosis of acute myocardial infarction (AMI) were excluded. We divided patients with troponin I elevation into three groups; no prescription, chronic prescription and new prescription of antithrombotic agents during ICU admission. We defined new prescription when patients were on antithrombotic agents, including antiplatelet agents, direct oral anticoagulants, and warfarin after troponin I was found to be elevated at ICU admission and chronic prescription, if antithrombotic agents were on medication list more than 30 days before ICU admission. Primary outcomes were 30-day and one-year all-cause mortality. Of 597 subjects who met inclusion criteria, 407 (68%) patients had elevated troponin I (>0.1 ng/mL) on ICU admission. These patients had increased 30-day [hazard ratio (HR), 1.679; 95% confidence interval (CI), 1.132–2.491; p = 0.009] and one-year (HR, 1.568; 95% CI, 1.180–2.083; p = 0.002) all-cause mortality compared with those without elevated troponin I. In patients with elevated troponin I, there was no significant difference of 30-day all-cause mortality among three groups (p = 0.051) whereas patients on chronic prescription showed significant survival benefit in one-year all-cause mortality when compared to those without or with new prescription (p= 0.008). .justify:after { content: ""; display:inline-block; width: 100%; } Conclusions: In critically ill patients, elevated troponin I in the absence of AMI was associated with poor prognosis. Newly prescribed antithrombotic agents in ICU didn’t reveal the difference in short and long-term prognosis while chronic antithrombotic agent use was associated with better one-year survival rate, suggesting that these drugs play a protective role in this high-risk population. .justify:after { content: ""; display:inline-block; width: 100%; } Keywords: critically ill patient, troponin I elevation, antithrombotic agent, intensive care unit .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Chuan-Tsai Tsai, Ya-Wen Lu, Ruey-Hsing Chou, Yi-Lin Tsai, Ming-Ren Kuo, Jiun-Yu Guo, Chi-Ting Lu, Chin-Sung Kuo, Po-Hsun Huang 2020. Associations of antithrombotic agent use with clinical outcomes in critically ill patients with troponin I elevation in the absence of acute coronary syndrome. protocols.io https://dx.doi.org/10.17504/protocols.io.bfbdjii6 Copy   

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Authors: Lucia Leierer, Antonio V. Herrera-Herrera, Margarita Jambrina-Enríquez
Group: Paleochar ERC
Summary: This is a protocol of a method used to obtain the total lipid extract (TLE) from archaeological sediment samples and the further elution of the TLE into different fraction and their preparation for GC-MS analysis.

Proper citation: Lucia Leierer, Antonio V. Herrera-Herrera, Margarita Jambrina-Enríquez 2019. Lipid Biomarker Extraction and Elution into different Fractions from sediment. protocols.io https://dx.doi.org/10.17504/protocols.io.yg2ftye Copy   

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Authors: M. T. Hirschauer, W. Neser, K. Wolter
Summary: VulPro is a vulture conservation organization in South Africa which has contributed to vulture research and rehabilitation since 2007. The organization’s efforts focus on captive breeding, rehabilitation, and research of the Cape vulture (Gyps coprotheres). The paper describes in detail VulPro’s methods for mass capturing using a specially designed walk-in trap, handling, weighing, collecting blood samples, and fitting patagial tags and tracking devices on vulture species. These protocols were employed with four species of southern African vultures: Cape vulture (Gyps coprotheres), African White-backed vulture (Gyps africanus), Lappet-faced vulture (Torgos tracheliotos), and Hooded vulture (Necrosyrtes monachus), but may be applied with other similar species.

Proper citation: M. T. Hirschauer, W. Neser, K. Wolter 2020. Protocol for mass capturing, handling, and fitting tracking devices and patagial tags on vultures. protocols.io https://dx.doi.org/10.17504/protocols.io.bmapk2dn Copy   

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Authors: Diep Ganguly
Group: Pogson Genomics Group
Summary: This protocol (based on the BigDye® Terminator v3.1 Cycle Sequencing Kit) is for performing terminator cycling sequencing reactions for Sanger sequencing of amplified PCR products or plasmid DNA on the 3130X genetic analyser (Applied Biosystems).

Proper citation: Diep Ganguly 2020. Dye-terminator DNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bhxkj7kw Copy   

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Authors: Dr. Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: March, 2012; based on sample preparation guide v2, catalog # RS-930-1021, part # 15026495 Rev. A, August 2011Modifications: samples in PCR tubes (because a plate/seal will not be used, some numbered steps are skipped); include two rounds of poly-A selection (with two separate sets of beads); repeat clean-up step after PCR to remove additional adapters; elute with 1.5 ul less volume off of beads (to leave 1 ul behind, not 2.5 ul)

Proper citation: Dr. Daniel Richter 2017. TruSeq RNA Preparation with Modification for Four Rounds of Poly-A Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.imucc6w Copy   

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Authors: Anthony Underwood

Proper citation: Anthony Underwood 2021. Navigating in UNIX. protocols.io https:// Copy   

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Authors: Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes

Proper citation: Binnypreet Kaur1, 2 , Drahomíra Faktorová1, 2, Priscila Peña-Diaz1 and Julius Lukeš1, 2 2019. Transfection of Diplonemids using AMAXA Nucleofactor apparatus. protocols.io https://dx.doi.org/10.17504/protocols.io.hf2b3qe Copy   

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Authors: José Carlos Serufo Filho, Denise Utsch Gonçalves, Zilma Reis
Summary: Background: The importance of effective communication for patient safety is critical when it comes to the practice of Doctor's handovers. Effective handovers are vital to patient safety and continuity of care, and this is Electronic handover provides better continuity of care than paper-based handover, and redistribution of tasks during working hours would benefit night staff. .justify:after { content: ""; display:inline-block; width: 100%; } Objective: To present the protocol used for the analysis of the quality of information registered in the medical document used in the transference of care sessions between doctors shifts. .justify:after { content: ""; display:inline-block; width: 100%; } Methods: This study is a clinical trial, controlled before-and-after in which observations were made before and after the implementation of an intervention, both in a group that receives the intervention. Handover session of physicians in the MATERNITY OF Hospital das Clínicas of the Universidade Federal de Minas Gerais, Brazil, randomly selected for monitoring. Eighteen before intervention and 18 after the intervention. .justify:after { content: ""; display:inline-block; width: 100%; } Results: It is expected with the application of the protocols and the progress of the research that we can evaluate and infer about the use of a computerized tool to carry out the shift, improve the quality of information, length of the transfer, traceability, and audit, and especially safety and quality in patient care. .justify:after { content: ""; display:inline-block; width: 100%; } Trial Registration: WHO: U1111-1211-4177

Proper citation: José Carlos Serufo Filho, Denise Utsch Gonçalves, Zilma Reis 2019. Electronic Record for Continuity of Patient Care: A use case for Doctor's handovers, in a tertiary maternity. protocols.io https://dx.doi.org/10.17504/protocols.io.vq7e5zn Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Heavy water can be used as a tracer for estimating metabolic rates (in vivo, in vitro) such as total energy expenditure (TEE) and fractional synthesis rates (FSR; eg. protein, lipids, triglycerides). When using both ²H²O and H²18O (DLW), TEE is estimated from the total production of CO² as measured by the differences in decay rates of labeled the 18O and ²H in body water over time following a single bolus of DLW (1). ²H²O can be used to estimate fractional synthesis rates of metabolic reactions such as those associated with proteins, lipids, triglycerides, and cholesterol (2,4).References: 1. Gas chromatography-mass spectrometry assay of the (18) O enrichment of water as trimethyl phosphate. Brunengraber DZ, McCabe BJ, Katanik J, and Previs SF. Anal Biochem 306: 278– 282 (2002). 2. Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis. Fang D, West RH, Manson ME, Ruddy J, Jiang D, Previs SF, Sonawane ND, Burgess JD, Kelley TJ.Respir Res.; 11:61 (2010). 3. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem.; 284(10):6101-8 (2009). 4. Novel application of the "doubly labeled" water method: measuring CO2 production and the tissue-specific dynamics of lipid and protein in vivo. Bederman IR, Dufner DA, Alexander JC, Previs SF. Am J Physiol Endocrinol Metab.; 290(5):E1048-56 (2006).

Proper citation: Henri Brunengraber 2019. Case - Heavy Water Assays by GC-mass spectrometry. protocols.io https://dx.doi.org/10.17504/protocols.io.ydsfs6e Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Summary: For use in Wet-mount Method for Enumeration of Aquatic Viruses

Proper citation: Matthew Sullivan 2016. Working Bead Solution. protocols.io https://dx.doi.org/10.17504/protocols.io.c84zyv Copy   

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Authors: Joao Vitor Molino

Proper citation: Joao Vitor Molino 2020. TAP agar plate preparation. protocols.io https:// Copy   

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Authors: James Fallon, Sophie Payne
Group: SPARC
Summary: Recording electrically-evoked compound action potentials (ECAPs) is performed to confirm that the electrode array is appropriately placed along the nerve. Recording ECAPS also allows confirmation that electrical stimulation is above neural threshold i.e. that electrical stimulation activates neural activity. The procedure is performed under anesthesia (immediately post-surgery) or in awake animals, and the procedure should incorporate all local requirements for standards of animal experimentation.

Proper citation: James Fallon, Sophie Payne 2020. Electrophysiological recording of electrically-evoked compound action potentials. protocols.io https://dx.doi.org/10.17504/protocols.io.bfwyjpfw Copy   

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Authors: Angel Justiz-Vaillant, Belkis Ferrer-Cosme
Group: University of the West Indies, [email protected]
Summary: IgA appears in 2 different molecular structures: monomeric (serum) and dimeric structure (secretory). The serum IgA has a molecular weight of 160 Kd and a serum concentration of 3 mg/mL. Secretory IgA (sIgA) has a molecular weight of 385 Kd and a mean serum concentration of 0.05 mg/mL. It appears in mucosa membranes as a dimer (with J chain when secreted) and protects the epithelial surfaces of the respiratory, digestive, and genitourinary system. IgA possesses a secretory component that prevents its enzymatic digestion. It activates the alternative pathway of activation of the complement system. [1]

Proper citation: Angel Justiz-Vaillant, Belkis Ferrer-Cosme 2020. ELISA for quantification of human immunoglobulin A (IgA) in serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj7vkrn6 Copy   

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Authors: Hung Liang Pai, Cheng-Ruei Yang

Proper citation: Hung Liang Pai, Cheng-Ruei Yang 2020. Specificity test of toehold. protocols.io https:// Copy   

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Authors: Emily Rosowski
Summary: Stains zebrafish larvae with Sudan Black B, which marks neutrophil granules.

Proper citation: Emily Rosowski 2018. Sudan Black staining of zebrafish larvae. protocols.io https://dx.doi.org/10.17504/protocols.io.rced2te Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. [1]The immunoregulatory cytokine IL-41 (also known as meteorin-like protein) is expressed at high levels in the synovium of patients with psoriatic arthritis (PsA).[2]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.2.Onuora S. Novel cytokine, IL-41, linked with PsA.Nat Rev Rheumatol. 2019;15(11):636. doi:10.1038/s41584-019-0314-7

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-41 in human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkeuktew Copy   

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Authors: Gloria Patricia Barrera1, Laura Fernanda Villamizar4, Carlos Espinel1, Edgar Mauricio Quintero2, Mariano Nicolás Belaich3, Deisy Liseth Toloza1, Pablo Daniel Ghiringhelli 3*, German Vargas2
Summary: We propose a Single-Strand Conformation Polymorphism (SSCP) methodology to recognize each species as an effective tool in Diatraea species identification.

Proper citation: Gloria Patricia Barrera1, Laura Fernanda Villamizar4, Carlos Espinel1, Edgar Mauricio Quintero2, Mariano Nicolás Belaich3, Deisy Liseth Toloza1, Pablo Daniel Ghiringhelli 3*, German Vargas2 2017. Single-Strand Conformation Polymorphism (SSCP) for Diatraea species identification. protocols.io https://dx.doi.org/10.17504/protocols.io.invcde6 Copy   

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Authors: Andreea S
Group: iGEM Groningen 2020
Summary: Neuropeptide-like proteins (NLPs) are a subclass of neuropeptides utilized by the nervous system of the nematodes. Altering the levels of the NLPs affects the behaviour of the neuronal networks in the nematode leading to a changed behavioural output. With this experiment, the behaviour of the Globodera pallida towards the root exudate of the potato plant is studied in a simple agar plate experiment. This chemosensory experiment is retrieved from the paper of Warnock and colleagues from 2017.

Proper citation: Andreea S 2020. Chemosensory assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bjwckpaw Copy   

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