Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10-8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

Recent advances in novel heterocyclic scaffolds for the treatment of drug-resistant malaria.

Malaria is a major public health problem all over the world, particularly in tropical and subtropical countries due to the development of resistance and most deadly infection is caused by Plasmodium falciparum. There is a direct need for the discovery of new drugs with unique structures and mechanism of action to treat sensitive and drug-resistant strains of various plasmodia for radical cure of this disease. Traditional compounds such as quinine and related derivatives represent a major source for the development of new drugs. This review presents recent modifications of 4-aminoquinoline and 8-aminoquinolone rings as leads to novel active molecules which are under clinical trials. The review also encompasses the other heterocyclic compounds emerged as potential antimalarial agents with promising results such as acridinediones and acridinone analogues, pyridines and quinolones as antimalarials. Miscellaneous heterocyclics such as tetroxane derivatives, indole derivatives, imidazolopiperazine derivatives, biscationic choline-based compounds and polymer-linked combined antimalarial drugs are also discussed. At last brief introduction to heterocyclics in natural products is also reviewed. Most of them have been under clinical trials and found to be promising in the treatment of drug-resistant strains of Plasmodium and others can be explored for the same purpose.

FRET studies of a landscape of Lac repressor-mediated DNA loops.

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-ß-D-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.

Use of heavy water (D2O) in developing thermostable recombinant p26 protein based enzyme-linked immunosorbent assay for serodiagnosis of equine infectious anemia virus infection.

Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4 °C, 37 °C, 42 °C, and 45 °C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37 °C < 42 °C < 45 °C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45 °C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

Optimization and validation of indirect ELISA using truncated TssB protein for the serodiagnosis of glanders amongst equines.

OBJECTIVE: To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. MATERIALS AND METHODS: In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein-a type 6 secretory effector protein--were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum. RESULTS: In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively. CONCLUSIONS: The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

Piloting a Novel Computational Framework for Identifying Prosthesis-Specific Contributions to Gait Deviations.

This paper introduces a novel computational framework for evaluating above-knee prostheses, addressing a major challenge in gait deviation studies: distinguishing between prosthesis-specific and patient-specific contributions to gait deviations. This innovative approach utilizes three separate computational models to quantify the changes in gait dynamics necessary to achieve a set of ideal gait kinematics across different prosthesis designs. The pilot study presented here employs a simple two-dimensional swing-phase model to conceptually demonstrate how the outcomes of this three-model framework can assess the extent to which prosthesis design impacts a user's ability to replicate the dynamics of able-bodied gait. Furthermore, this framework offers potential for optimizing passive prosthetic devices for individual patients, thereby reducing the need for real-life experiments, clinic visits, and overcoming rehabilitation challenges.

Machine Learning Elucidates Electrophysiological Properties Predictive of Multi- and Single-Firing Human and Mouse Dorsal Root Ganglia Neurons.

Human and mouse dorsal root ganglia (hDRG and mDRG) neurons are important tools in understanding the molecular and electrophysiological mechanisms that underlie nociception and drive pain behaviors. One of the simplest differences in firing phenotypes is that neurons are single-firing (exhibit only one action potential) or multi-firing (exhibit 2 or more action potentials). To determine if single- and multi-firing hDRG neurons exhibit differences in intrinsic properties, firing phenotypes, and AP waveform properties, and if these properties could be used to predict multi-firing, we measured 22 electrophysiological properties by whole-cell patch-clamp electrophysiology of 94 hDRG neurons from six male and four female donors. We then analyzed the data using several machine learning models to determine if these properties could be used to predict multi-firing. We used 1,000 iterations of Monte Carlo cross-validation to split the data into different train and test sets and tested the logistic regression, k-nearest neighbors, random forest, support vector classifier, and XGBoost machine learning models. All models tested had a >80% accuracy on average, with support vector classifier, and XGBoost performing the best. We found that several properties correlated with multi-firing hDRG neurons and together could be used to predict multi-firing neurons in hDRG including a long decay time, a low rheobase, and long first spike latency. We also found that the hDRG models were able to predict multi-firing with 90% accuracy in mDRG neurons. Understanding these properties could be beneficial in the elucidation of targets on peripheral sensory neurons related to pain.

Machine learning elucidates electrophysiological properties predictive of multi- and single-firing human and mouse dorsal root ganglia neurons.

Human and mouse dorsal root ganglia (hDRG and mDRG) neurons are important tools in understanding the molecular and electrophysiological mechanisms that underlie nociception and drive pain behaviors. One of the simplest differences in firing phenotypes is that neurons are single-firing (exhibit only one action potential) or multi-firing (exhibit 2 or more action potentials). To determine if single- and multi-firing hDRG exhibit differences in intrinsic properties, firing phenotypes, and AP waveform properties, and if these properties could be used to predict multi-firing, we measured 22 electrophysiological properties by whole-cell patch-clamp electrophysiology of 94 hDRG neurons from 6 male and 4 female donors. We then analyzed the data using several machine learning models to determine if these properties could be used to predict multi-firing. We used 1000 iterations of Monte Carlo Cross Validation to split the data into different train and test sets and tested the Logistic Regression, k-Nearest Neighbors, Random Forest, Supported Vector Classification, and XGBoost machine learning models. All models tested had a greater than 80% accuracy on average, with Supported Vector Classification and XGBoost performing the best. We found that several properties correlated with multi-firing hDRG neurons and together could be used to predict multi-firing neurons in hDRG including a long decay time, a low rheobase, and long first spike latency. We also found that the hDRG models were able to predict multi-firing with 90% accuracy in mDRG. Targeting the neuronal properties that lead to multi-firing could elucidate better targets for treatment of chronic pain.

Electrophysiological Analyses of Human Dorsal Root Ganglia and Human Induced Pluripotent Stem Cell-derived Sensory Neurons From Male and Female Donors.

Human induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) and human dorsal root ganglia neurons (hDRG-N) are popular tools in the field of pain research; however, few groups make use of both approaches. For screening and analgesic validation purposes, important characterizations can be determined of the similarities and differences between hDRG-N and hiPSC-SNs. This study focuses specifically on the electrophysiology properties of hDRG-N in comparison to hiPSC-SNs. We also compared hDRG-N and hiPSC-SNs from both male and female donors to evaluate potential sex differences. We recorded neuronal size, rheobase, resting membrane potential, input resistance, and action potential waveform properties from 83 hiPSCs-SNs (2 donors) and 108 hDRG-N neurons (8 donors). We observed several statistically significant electrophysiological differences between hDRG-N and hiPSC-SNs, such as size, rheobase, input resistance, and several action potential waveform properties. Correlation analysis also revealed many properties that were positively or negatively correlated, some of which were differentially correlated between hDRG-N and hiPSC-SNs. This study shows several differences between hDRG-N and hiPSC-SNs and allows a better understanding of the advantages and disadvantages of both for use in pain research. We hope this study will be a valuable resource for pain researchers considering the use of these human in vitro systems for mechanistic studies and/or drug development projects. PERSPECTIVE: hiPSC-SNs and hDRG-N are popular tools in the field of pain research. This study allows for a better functional understanding of the pros and cons of both tools.

Guggulipid of Commiphora mukul, with antiallodynic and antihyperalgesic activities in both sciatic nerve and spinal nerve ligation models of neuropathic pain.

CONTEXT: Guggulipid is a neutral fraction of ethyl acetate extract of gum resin of the tree Commiphora mukul Engl. (Burseraceae) and used in Ayurvedic medicine for treatment of neurological disorders. OBJECTIVES: The present study was undertaken to assess the antiallodynic and antihyperalgesic activities of guggulipid in rats. MATERIALS AND METHODS: The screening study included the CCI and L5-L6 SNL models of neuropathic pain. Guggulipid (100 and 50 mg/kg) or saline was administered intraperitoneally in a blinded, randomized manner from postoperative day (POD) 7 to 13. Paw withdrawal duration (PWD) to spontaneous pain, chemical allodynia and mechanical hyperalgesia and paw withdrawal latency (PWL) to mechanical allodynia and thermal hyperalgesia were tested before surgery, before and after guggulipid or saline administration (from POD7 to 13) and after the withdrawal of treatment (from POD14 to 20). RESULTS: The activity profiles of the different doses of guggulipid were found to vary with time. In CCI rats, guggulipid (100 and 50 mg/kg) significantly (p < 0.05) reduced the spontaneous pain, mechanical allodynia and mechanical and thermal hyperalgesia responses and the LD50 of guggulipid was 1600 mg/kg. In SNL rats, both doses of guggulipid were found to be ineffective in reversing the spontaneous pain but showing antiallodynic and antihyperalgesic activity. DISCUSSION AND CONCLUSION: The results demonstrated that guggulipid produce antinociception in the peripheral nerve injury (CCI and SNL) models of neuropathic pain. The underlying mechanisms are expected to be modulating microglial activation occurring due to peripheral nerve injury.

A review on blockchain for DNA sequence: security issues, application in DNA classification, challenges and future trends.

In biological science, the study of DNA sequences is considered an important factor because it carries the genomic details that can be used by researchers and doctors for the early prediction of disease using DNA classification. The NCBI has the world's largest database of genetic sequences, but the security of this massive amount of data is currently the greatest issue. One of the options is to encrypt these genetic sequences using blockchain technology. As a result, this paper presents a survey on healthcare data breaches, the necessity for blockchain in healthcare, and the number of research studies done in this area. In addition, the report suggests DNA sequence classification for earlier disease identification and evaluates previous work in the field.

Rare Case of Remitting Seronegative Symmetrical Synovitis with Pitting Edema Syndrome with Monoclonal Gammopathy of Undetermined Significance.

BACKGROUND Remitting seronegative symmetrical synovitis with pitting edema (RS3PE) is a rare condition with underlying polyarthritis, pitting edema, and negative rheumatoid factor. It can be associated with an underlying rheumatological condition or can present as a paraneoplastic syndrome with malignancy. We present a rare case of RS3PE associated with monoclonal gammopathy of undermined significance (MGUS). CASE REPORT A 62-year-old man presented in ambulatory medicine clinic with 3-month swelling of distal lower extremities that progressed to distal upper extremities. He had pain and morning stiffness in hands, left elbow, and left shoulder. Examination revealed 3+ pitting edema in bilateral hands, feet, legs, and thighs. Laboratory studies revealed normal blood counts and renal and liver functions. Erythrocyte sedimentation rate was normal; C-reactive protein was mildly elevated (0.7 mg/dL). Echocardiogram and computed tomography of chest, abdomen, and pelvis revealed mild splenomegaly (14.5 cm). Serum protein electrophoresis revealed IgG kappa monoclonal peak of 0.1 g/dL. Beta-2 microglobulin was elevated (7.4 mg/L); LDH was elevated (264 U/L). No lytic lesions were present in bones. RS3PE was diagnosed based on established diagnostic criteria. Prednisone produced significant improvement in swelling within 72 h of start; however, he required a longer duration of steroid treatment due to relapse and continued periodic MGUS surveillance. CONCLUSIONS Our case highlights the importance of awareness of this condition in general practice to help with timely diagnosis and intervention, as this condition is steroid responsive. Also, it is important to screen for underlying autoimmune condition, hematological, and solid organ malignancies with appropriate workup.

Plant-derived natural products targeting ion channels for pain.

Chronic pain affects approximately one-fifth of people worldwide and reduces quality of life and in some cases, working ability. Ion channels expressed along nociceptive pathways affect neuronal excitability and as a result modulate pain experience. Several ion channels have been identified and investigated as potential targets for new medicines for the treatment of a variety of human diseases, including chronic pain. Voltage-gated channels Na+ and Ca2+ channels, K+ channels, transient receptor potential channels (TRP), purinergic (P2X) channels and acid-sensing ion channels (ASICs) are some examples of ion channels exhibiting altered function or expression in different chronic pain states. Pharmacological approaches are being developed to mitigate dysregulation of these channels as potential treatment options. Since natural compounds of plant origin exert promising biological and pharmacological properties and are believed to possess less adverse effects compared to synthetic drugs, they have been widely studied as treatments for chronic pain for their ability to alter the functional activity of ion channels. A literature review was conducted using Medline, Google Scholar and PubMed, resulted in listing 79 natural compounds/extracts that are reported to interact with ion channels as part of their analgesic mechanism of action. Most in vitro studies utilized electrophysiological techniques to study the effect of natural compounds on ion channels using primary cultures of dorsal root ganglia (DRG) neurons. In vivo studies concentrated on different pain models and were conducted mainly in mice and rats. Proceeding into clinical trials will require further study to develop new, potent and specific ion channel modulators of plant origin.

Electrophysiological analyses of human dorsal root ganglia and human induced pluripotent stem cell-derived sensory neurons from male and female donors.

Human induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) and human dorsal root ganglia (hDRG) neurons are popular tools in the field of pain research; however, few groups make use of both approaches. For screening and analgesic validation purposes, important characterizations can be determined of the similarities and differences between hDRG and hiPSC-SNs. This study focuses specifically on electrophysiology properties of hDRG in comparison to hiPSC-SNs. We also compared hDRG and hiPSC-SNs from both male and female donors to evaluate potential sex differences. We recorded neuronal size, rheobase, resting membrane potential, input resistance, and action potential waveform properties from 83 hiPSCs-SNs (2 donors) and 108 hDRG neurons (9 donors). We observed several statistically significant electrophysiological differences between hDRG and hiPSC-SNs, such as size, rheobase, input resistance, and several actional potential (AP) waveform properties. Correlation analysis also revealed many properties that were positively or negatively correlated, some of which were differentially correlated between hDRG and hiPSC-SNs. This study shows several differences between hDRG and hiPSC-SNs and allows better understanding of the advantages and disadvantages of both for use in pain research. We hope this study will be a valuable resource for pain researchers considering the use of these human in vitro systems for mechanistic studies and/or drug development projects.

Antiviral activity of molnupiravir against COVID-19: a schematic review of evidences.

Background: The study was aimed at encapsulating the evidence of in vitro and in vivo antiviral activities of molnupiravir and its active form against highly pathogenic SARS-CoV-2, the pathogen responsible for COVID-19, and finding out the efficacy and safety of molnupiravir in clinical trials. Main body: Information on publications was explored on several databases, gray literature was reviewed, and the outcomes were discussed narratively. Molnupiravir's antiviral efficacy and associated mechanism of action have been verified in vitro against both non-COVID and multiple coronaviruses. Molnupiravir has been tried in preclinical investigations in numerous animal models against non-coronaviruses. Clinical studies in several countries are now being conducted to evaluate its antiviral efficacy in persons infected with COVID-19. The medication displays antiviral effect via generation of copying mistakes during viral RNA replication. Conclusions: Molnupiravir is the first oral antiviral medicine to show considerable and convincing antiviral activity in vitro and in animal models. Molnupiravir stops the spread of SARS-CoV-2 in animals that have been infected and in cells grown in a lab. In a clinical research, early molnupiravir treatment reduced hospitalization and death risk in unvaccinated individuals with COVID-19. In the battle against SARS-CoV-2, it could be a potent weapon. However, its role in COVID-19 in moderate to severe cases is still up in the air, and more research is needed.

A comprehensive tool for rapid and accurate prediction of disease using DNA sequence classifier.

In the current pandemic situation where the coronavirus is spreading very fast that can jump from one human to another. Along with this, there are millions of viruses for example Ebola, SARS, etc. that can spread as fast as the coronavirus due to the mobilization and globalization of the population and are equally deadly. Earlier identification of these viruses can prevent the outbreaks that we are facing currently as well as can help in the earlier designing of drugs. Identification of disease at a prior stage can be achieved through DNA sequence classification as DNA carries most of the genetic information about organisms. This is the reason why the classification of DNA sequences plays an important role in computational biology. This paper has presented a solution in which samples collected from NCBI are used for the classification of DNA sequences. DNA sequence classification will in turn gives the pattern of various diseases; these patterns are then compared with the samples of a newly infected person and can help in the earlier identification of disease. However, feature extraction always remains a big issue. In this paper, a machine learning-based classifier and a new technique for extracting features from DNA sequences based on a hot vector matrix have been proposed. In the hot vector representation of the DNA sequence, each pair of the word is represented using a binary matrix which represents the position of each nucleotide in the DNA sequence. The resultant matrix is then given as an input to the traditional CNN for feature extraction. The results of the proposed method have been compared with 5 well-known classifiers namely Convolution neural network (CNN), Support Vector Machines (SVM), K-Nearest Neighbor (KNN) algorithm, Decision Trees, Recurrent Neural Networks (RNN) on several parameters including precision rate and accuracy and the result shows that the proposed method gives an accuracy of 93.9%, which is highest compared to other classifiers.

Use of recombinant calflagin protein as a potential candidate for diagnosis of Trypanosoma evansi infection.

Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency.

Manual toothbrushing reinforced with audiovisual instruction versus powered toothbrushing among institutionalized mentally challenged subjects--a randomized cross-over clinical trial.

AIM: To assess and compare the effectiveness of Manual toothbrushing reinforced with audiovisual instructions with powered toothbrushing, among the institutionalized mentally challenged individuals under supervision of trained caretakers. MATERIALS AND METHODS: A randomized cross over clinical trial of 6 months duration which included 16 subjects consisting of two phases of three months, for each of the 2 groups. In group A subjects were given manual toothbrushes with audio-visual aid followed by the powered toothbrushes & vice versa for group B. All subjects were instructed by trained care takers. An evaluation of the plaque & gingival scores was done at the end of 1, 2 & 3 months for both the groups. RESULTS: Phase I showed statistically significant decrease in mean plaque scores (p=0.037) but insignificant mean gingival scores (p=0.189) in group A at end of 3 months. In phase II, statistically insignificant decrease in mean plaque & gingival scores were recorded at end of 3 months. In group B a statistically significant decrease in both plaque (p=0.002) & gingival (p=0.001) was found at end in both phases. Comparison of mean plaque & gingival scores of manual & powered toothbrushes at different intervals in both groups were statistically insignificant. CONCLUSION: For mentally challenged individuals, manual toothbrushes reinforced with audio-visual instructions for brushing may be comparable to the use of powered toothbrushes.

Evidence of carrier mediated transport of ascorbic acid through mammalian cornea.

The purpose of the present study was to evaluate the transport of ascorbic acid, a water soluble molecule, through a predominantly lipophilic cornea. Thus in-vitro permeation of ascorbic acid from aqueous drops through freshly excised mammalian cornea was studied. Aqueous isotonic ophthalmic solutions of ascorbic acid of different concentrations (0.125% w/v to 2% w/v) (pH 5.4) were made. Further 1.0% w/v or 0.5% w/v ascorbic acid solution containing NaCl or dextrose as tonicity modifiers or Na(+)K(+)-ATPase inhibitors were also made. Permeation characteristics of drug were evaluated by putting 1 ml formulation on freshly excised cornea fixed between donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring the drug permeated in the receptor by spectrophotometry at 265 nm, after 120 min. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Dunnett's test or paired t-test. Increase in drug concentration in the formulation resulted in an increase in the quantity permeated but after a certain level increase in permeation with increase in concentration was minimal. Aqueous drops made isotonic with dextrose showed decreased permeation through paired cornea compared with aqueous drops made isotonic with NaCl from 1% w/v ascorbic acid solution suggesting likely involvement of Na(+) co-transporter but there was decreased permeation through 0.5% w/v ascorbic acid solution made isotonic with NaCl as compared to solution made isotonic with dextrose. Further aqueous drops containing Na(+)K(+)-ATPase inhibitor {MAG-Mono Ammonium Glycyrrhizinate (25 µmol)} showed decreased corneal permeation from 0.5% w/v ascorbic acid solution but there was not significant decrease from 1% ascorbic acid solution since MAG is a competitive inhibitor of ascorbic acid. Aqueous drops containing Na(+)K(+)-ATPase inhibitor {MAG (50 µmol) or Ouabain (1 mmol)} showed decreased corneal permeation of ascorbic acid compared with control from 1% ascorbic acid solution confirming the involvement of Na(+) co-transporter.

Post-acute COVID-19 functional movement disorder.

Movement disorders are rare compared to other neurological manifestations of COVID-19. Patients who have recovered from acute severe acute respiratory syndrome coronavirus-2 infection continue to have multiple debilitating symptoms months later. We report a case of 54-year-old man who presented with repetitive flexion movement of head which started 2 months after severe acute respiratory syndrome coronavirus-2 infection. Extensive work-up including neurological examination, neuroimaging, cerebrospinal fluid analysis, and electroencephalogram were normal. The self-reported questionnaires for depression and anxiety were suggestive of severe anxiety and depression. The patient continued to have the jerky movements besides cognitive impairment, frequent headaches, intermittent shortness of breath, sleeping difficulties, fatigue, and dizziness at 1-year follow-up. This case highlights the presentation of functional movement disorder as one of the manifestations of underlying neuropsychiatric condition. Our patient had significant effect on quality of life with high symptom burden which further highlights the struggle and unmet needs of the patients with multiple symptoms after severe acute respiratory syndrome coronavirus-2 infection.