Polymerase Chain Reaction Based-Assays for Rapid Detection and Subtyping Of Type a Influenza Viruses

Highly pathogenic avian influenza (HPAI) virus causes high morbidity, mortality and still is a big threat in poultry industry. The recently raised awareness of the threat of a new influenza pandemic has stimulated interests in the development of a rapid detection method for influenza A viruses. In this study, four diagnostic methods for the detection of type A influenza viruses were explored. A conventional one-tube nucleoprotein reverse transcriptase polymerase chain reaction (NP RT-PCR) was developed for rapid detection of avian influenza A viruses. This method successfully detected 14 different haemagglutinin (HA) subtypes of different origins. A multiplex RT-PCR that successfully amplified three RNA templates of H5, H7 and H9 in one tube was also developed. The designed primers were specific in amplification of the HA genes of H5, H7 and H9 of type A influenza viruses. No amplification was observed with other avian infectious viruses such as Newcastle disease virus, infectious bronchitis virus and infectious bursa1 disease virus. An enzyme-linked immunosorbent assay (ELISA) detection method was then developed to detect the amplified PCR products. This method was 10 times more sensitive than the detection of PCR product using agarose gel electrophoresis. This method (RT-PCR-ELISA) was as sensitive as virus isolation in specific-pathogen-free (SPF) embryonated eggs. The detection limit of the RT-PCR-ELISA was compared with agarose gel electrophoresis and one-step SYBR Green I real time PCR. The RTPCR- ELISA was 10 times less sensitive than SYBR Green I real time PCR. The whole process for the detection of type A influenza virus and the avian H5, H7 and H9 subtypes, from extraction of RNA to analysis of PCR product by agarose gel electrophoresis or colorimetric assay can be completed within 6 h. It provides a rapid means of identification of the type and subtypes of influenza viruses and would be very useful for their surveillance. The advantage of using an ELSA reader is in removing any element of subjective interpretation as a source of error. The methods developed in this study, were tested on suspected cases. The finding indicated that the methods are rapid, sensitive and specific, and thus would be a method of choice for the surveillance of avian influenza virus. Moreover, the RT-PCR-ELISA method allows handling of a large number of samples and can be used in many diagnostic laboratories. Among the four methods developed, the SYBR Green I real time PCR was the best method in terms of sensitivity and specificity. This is followed by RT-PCR-ELSA, multiplex and conventional RT-PCR assays

Molecular Movement of the Voltage Sensor in a K Channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 Å) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 Å) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model

Liability of a Surgeon for the Extension of an Authorized Operation

Polyunsaturated fatty acids modulate the voltage dependence of several voltage-gated ion channels, thereby being potent modifiers of cellular excitability. Detailed knowledge of this molecular mechanism can be used in designing a new class of small-molecule compounds against hyperexcitability diseases. Here, we show that arginines on one side of the helical K-channel voltage sensor S4 increased the sensitivity to docosahexaenoic acid (DHA), whereas arginines on the opposing side decreased this sensitivity. Glutamates had opposite effects. In addition, a positively charged DHA-like molecule, arachidonyl amine, had opposite effects to the negatively charged DHA. This suggests that S4 rotates to open the channel and that DHA electrostatically affects this rotation. A channel with arginines in positions 356, 359, and 362 was extremely sensitive to DHA: 70 mu M DHA at pH 9.0 increased the current greater than500 times at negative voltages compared with wild type (WT). The small-molecule compound pimaric acid, a novel Shaker channel opener, opened the WT channel. The 356R/359R/362R channel drastically increased this effect, suggesting it to be instrumental in future drug screening

Detection of Newcastle Disease, H9N2 Avian Influenza, and Infectious Bronchitis Viruses in Respiratory Diseases in Backyard Chickens in Ahvaz, Iran, in 2014-2015

Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious bronchitis virus (IBV) are the most prevalent viral pathogens in the Iranian poultry industry. This study aimed to reveal the presence of these viruses in the backyard chickens in Ahvaz, located in the Southwest of Iran. A total of 100 chickens with respiratory signs and mortality were examined by taking the blood samples as well as tracheal and cloacal swabs. Most of the chickens had not received any vaccine. The blood samples were assessed for the antibodies against NDV and AIV by haemagglutination inhibition test, and against IBV by enzyme-linked immunosorbent assay. The swab samples were utilized for molecular detection using reverse transcription polymerase chain reaction (RT-PCR). Based on the results of the serologic test, 77%, 45%, and 38.4% of the birds were positive for NDV, AIV, and IBV, respectively. In the RT-PCR, 95% of the birds were positive for one of the three viruses. The detection rates of NDV, AIV, and IBV were 60%, 34%, and 55%, respectively. The coinfections of AIV/NDV, AIV/IBV, NDV/IBV, and AIV/NDV/ IBV were observed in 13%, 4%, 23%, and 7% of the sampled chickens, respectively. The results demonstrated that the Iranian backyard chickens were infected with NDV, AIV, and IBV. This could pose a threat to the commercial poultry; therefore, preventive measures need to be implemented in this regard

An electrostatic potassium channel opener targeting the final voltage sensor transition

Free polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of voltage-gated ion channels. As an important consequence thereof, PUFAs can suppress epileptic seizures and cardiac arrhythmia. However, molecular details for the interaction between PUFA and ion channels are not well understood. In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect. Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect. Because different voltage-gated K channels have different charge profiles, this implies channel-specific PUFA effects. The identified site and the pharmacological mechanism will potentially be very useful in future drug design of small-molecule compounds specifically targeting neuronal and cardiac excitability

Oxaliplatin neurotoxicity – no general ion channel surface-charge effect

<p>Abstract</p> <p>Background</p> <p>Oxaliplatin is a platinum-based chemotherapeutic drug. Neurotoxicity is the dose-limiting side effect. Previous investigations have reported that acute neurotoxicity could be mediated via voltage-gated ion channels. A possible mechanism for some of the effects is a modification of surface charges around the ion channel, either because of chelation of extracellular Ca²⁺, or because of binding of a charged biotransformation product of oxaliplatin to the channel. To elucidate the molecular mechanism, we investigated the effects of oxaliplatin and its chloride complex [Pt(dach)oxCl]^-on the voltage-gated Shaker K channel expressed in <it>Xenopus </it>oocytes. The recordings were made with the two-electrode and the cut-open oocyte voltage clamp techniques.</p> <p>Conclusion</p> <p>To our surprise, we did not see any effects on the current amplitudes, on the current time courses, or on the voltage dependence of the Shaker wild-type channel. Oxaliplatin is expected to bind to cysteines. Therefore, we explored if there could be a specific effect on single (E418C) and double-cysteine (R362C/F416C) mutated Shaker channels previously shown to be sensitive to cysteine-specific reagents. Neither of these channels were affected by oxaliplatin. The clear lack of effect on the Shaker K channel suggests that oxaliplatin or its monochloro complex has no general surface-charge effect on the channels, as has been suggested before, but rather a specific effect to the channels previously shown to be affected.</p

Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening

In ether-à-go-go (eag) K+ channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ∼4 Å apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg2+. In I287D+F324D, Mg2+ significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg2+ binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg2+ shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg2+ binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation

Review on the Oncology Practice in the Midst of COVID-19 Crisis: The Challenges and Solutions

As of late 2019, the outbreak of novel coronavirus disease (COVID-19) that started in China has rapidly afflicted all over the world. The COVID-19 pandemic has challenged health-care facilities to provide optimal care. In this context, cancer care requires special attention because of its peculiar status by including patients who are commonly immunocompromised and treatments that are often highly toxic. In this review article, we have classified the main impacts of the COVID-19 pandemic on oncology practices followed by their solutions into ten categories, including impacts on (1) health care providers, (2) medical equipment, (3) access to medications, (4) treatment approaches, (5) patients� referral, (6) patients� accommodation, (7) patients� psychological health, (8) cancer research, (9) tumor board meetings, and (10) economic income of cancer centers. The effective identification and management of all these challenges will improve the standards of cancer care over the viral pandemic and can be a practical paradigm for possible future crises. © 2021. All rights reserved

Migraine headache in Multiple Sclerosis. Is more frequent among MS patients?

Objectives. Multiple sclerosis (MS) is a chronic disease of the central nervous system that is associated with inflammation, demyelination of neurons and gliosis. There are different reports about the association between migraine and MS. Taking note of headaches experienced by people with MS and providing appropriate treatment can help enhance their quality of life. We aimed to determine the frequency of migraine headache in MS patients. Materials and methods. This was a descriptive cross-sectional study, conducted in 2020 among 125 MS patients referred to neurology clinic of Poursina Hospital or registered in Guilan MS Registry System and 125 controls with minor head trauma in Rasht. After obtaining written consent, all the subjects were asked to fulfil a checklist about their demographics, MS and headache characteristics by phone call. Outcomes. In this study 96 RRMS, 19 PPMS and 10 SPMS patients participated among which frequency of mild, moderate and severe disability was 74.4%, 22.4% and 3.2%, respectively. The frequency of migraine in MS patients was significantly higher than the control group [28.8% (n=36) vs. 12.8% (n=16) p=0.002]. After adjusting the confounding factors a significant relationship was found between MS and migraine (OR: 2.76, p = 0.004). Conclusions. MS patients experience migraine headaches approximately twice the general population

Detecting acute neurotoxicity during platinum chemotherapy by neurophysiological assessment of motor nerve hyperexcitability

<p>Abstract</p> <p>Background</p> <p>Platinum-based drugs, such as cisplatin and oxaliplatin, are well-known for inducing chronic sensory neuropathies but their acute and motor neurotoxicities are less well characterised. Use was made of nerve conduction studies and needle electromyography (EMG) to assess motor nerve excitability in cancer patients during their first treatment cycle with platinum-based chemotherapy in this study.</p> <p>Methods</p> <p>Twenty-nine adult cancer patients had a neurophysiological assessment either before oxaliplatin plus capecitabine, on days 2 to 4 or 14 to 20 after oxaliplatin plus capecitabine, or on days 2 to 4 after carboplatin plus paclitaxel or cisplatin, undertaken by a neurophysiologist who was blinded to patient and treatment details. Patients completed a symptom questionnaire at the end of the treatment cycle.</p> <p>Results</p> <p>Abnormal spontaneous high frequency motor fibre action potentials were detected in 100% of patients (n = 6) and 72% of muscles (n = 22) on days 2 to 4 post-oxaliplatin, and in 25% of patients (n = 8) and 13% of muscles (n = 32) on days 14 to 20 post-oxaliplatin, but in none of the patients (n = 14) or muscles (n = 56) tested prior to oxaliplatin or on days 2 to 4 after carboplatin plus paclitaxel or cisplatin. Repetitive compound motor action potentials were less sensitive and less specific than spontaneous high frequency motor fibre action potentials for detection of acute oxaliplatin-induced motor nerve hyperexcitability but were present in 71% of patients (n = 7) and 32% of muscles (n = 32) on days 2 to 4 after oxaliplatin treatment. Acute neurotoxicity symptoms, most commonly cold-induced paraesthesiae and jaw or throat tightness, were reported by all patients treated with oxaliplatin (n = 22) and none of those treated with carboplatin plus paclitaxel or cisplatin (n = 6).</p> <p>Conclusions</p> <p>Abnormal spontaneous high frequency motor fibre activity is a sensitive and specific endpoint of acute oxaliplatin-induced motor nerve hyperexcitability, detectable on EMG on days 2 to 4 post-treatment. Objective EMG assessment of motor nerve excitability could compliment patient-reported symptomatic endpoints of acute oxaliplatin-induced neurotoxicity in future studies.</p