Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay orpolymerase chain reaction

Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS)virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein,presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDSvirus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared withthe other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbentassay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum againstEDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome.Following experimental infection of oestrogen-treated chickens with EDS virus, cloaca1 swabs, oviduct, uterusand spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly andafter a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by ACELISAor PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommendeddue to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommendthat PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged inembryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.     

Evaluation of Micronuclei and Cytomorphometric Changes in Patients with Different Tobacco Related Habits Using Exfoliated Buccal Cells

Background: Tobacco is one of the main reasons behind the occurrence of oral cancer. Oral cancer, even though being the tenth most common cancer in the world, gets diagnosed at an advanced stage and ends up with poor prognosis. So early diagnosis is the need of the hour. Our study aimed to evaluate the genotoxic changes in patients with different tobacco habits using buccal exfoliated cells. Methods: Buccal smears were taken from smokers (30), smokeless tobacco users (30), combined tobacco users (30) and controls (30) with clinically normal oral mucosa. All the smears were stained with Papanicolaou stain and Feulgen stain and viewed under light microscope for the evaluation of mean number of micronuclei, mean micronuclei per cell, frequency of cells showing micronuclei, nuclear area, cytoplasmic area, nuclear-cytoplasmic ratio. Results: Mean number of micronuclei, mean micronuclei per cell, frequency of cells showing micronuclei, and nuclear area were significantly increased in tobacco users than controls, especially in combined tobacco users. Nuclear-cytoplasmic ratio was increased and cytoplasmic area was decreased in tobacco users than controls. Conclusion: Tobacco in any consumable form is genotoxic. Smoking and smokeless tobacco, when consumed together, synergistically causes higher genetic damage. Different tobacco habits have different deleterious effects on oral mucosa, and these effects are more pronounced when the patients have combined habits. So, detecting the genotoxic changes through exfoliative cytology can be used as a simple yet reliable marker for early detection of carcinogenesis.     

Implicit Bias in Pediatric Academic Medicine

Objective

Despite known benefits of diversity, certain racial/ethnic groups remain underrepresented in academic pediatrics. Little research exists regarding unconscious racial attitudes among pediatric faculty responsible for decisions on workforce recruitment and retention in academia. This study sought to describe levels of unconscious racial bias and perceived barriers to minority recruitment and retention among academic pediatric faculty leaders.

Implausibility of the vibrational theory of olfaction

The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. A previous study reported that human subjects differentiated hydrogen/deuterium isotopomers (isomers with isotopic atoms) of the musk compound cyclopentadecanone as evidence supporting the theory. Here, we find no evidence for such differentiation at the molecular level. In fact, we find that the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR expression system and robustly responding to cyclopentadecanone and muscone, fails to distinguish isotopomers of these compounds in vitro. Furthermore, the mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other selected human and mouse ORs, responded similarly to normal, deuterated, and ¹³C isotopomers of their respective ligands, paralleling our results with the musk receptor OR5AN1. These findings suggest that the proposed vibration theory does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3, or other ORs examined. Also, contrary to the vibration theory predictions, muscone-d₃₀ lacks the 1,380- to 1,550-cm⁻¹ IR bands claimed to be essential for musk odor. Furthermore, our theoretical analysis shows that the proposed electron transfer mechanism of the vibrational frequencies of odorants could be easily suppressed by quantum effects of nonodorant molecular vibrational modes. These and other concerns about electron transfer at ORs, together with our extensive experimental data, argue against the plausibility of the vibration theory.In 1870, the British physician William Ogle wrote: “As in the eye and the ear the sensory impression is known to result not from the contact of material particles given off by the object seen or heard, but from waves or undulations of the ether or the air, one cannot but suspect that the same may be true in the remaining sense, and that the undulatory theory of smell… [may be] the true one” (1, 2). Of the 29 different “theories of odour” listed in the 1967 edition of The Chemical Senses (3), nine associate odor with vibrations, particularly those theories championed by Dyson (4, 5) and Wright (6–8). However, the premise that olfaction involves detection of vibrational frequencies of odorants remains highly speculative because neither the structures of the odorant receptors (ORs) nor the binding sites or the activation mechanisms triggered upon odorant binding to ORs have been established. In 1996–1997, Turin (9–12) elaborated on the undulatory theory of smell, as considered in more detail below, and suggested that a mechanism analogous to inelastic electron tunneling spectroscopy (13) may be involved, where tunneling electrons in the receptor probe the vibrational frequencies of odorants. In 2013, Gane et al. (14) commented that “whether olfaction recognizes odorants by their shape, their molecular vibrations, or both remains an open and controversial question” and that “a convenient way to address [this question] is to test for odor character differences between deuterated and nondeuterated odorant isotopomers since these have identical ground-state conformations but different vibrational modes.” Gane et al. (14) also stated that a particularly appropriate test case would involve odorants containing “more CH group… [such as] musks [which] are among the largest odorants and typically contain 15–18 carbons and 28 or more hydrogens.”In judging the plausibility of the vibration theory, we use a multipronged approach:

• i)We consider the concepts of shape vs. vibration theory and odorant perception vs. reception.
• ii)As a test of the vibration theory, we have prepared a series of isotopomers of musks and other compounds, containing up to 30 C–H or C–D bonds as test odorants, which are evaluated using in vitro activation of receptors identified by us and other groups as being highly responsive to these isotopomers.
• iii)We consider the confounding effects of impurities and isotope effects in interpreting odorant perception, as well as the validity of requirements for specific IR bands for recognition of musks by their receptors.
• iv)We examine the physical validity of the models developed to support the vibration theory.
• v)We consider the specific limitations of our in vitro approach using isotopomers to evaluate the vibration theory, based primarily on results obtained with a single identified human musk OR, in addition to other OR/ligand pairs.
• vi)We consider plausible nonvibration theory models for docking of musks to the human musk receptor, OR5AN1, where the musk carbonyl group functions as a hydrogen bond acceptor.

Gane et al. (14) have framed the argument for olfactory discrimination of hydrogen isotopomers as one of “shape” vs. “vibration.” However, neither the binding modes of isotopomers nor their activation mechanisms are known. ORs belong to the superfamily of class A G protein-coupled receptors (GPCRs), which are known to be activated through allosteric conformational changes induced upon ligand binding even without triggering any kind of electron transfer processes. Ligand–receptor interactions can be both attractive and repulsive, involving hydrogen bonding, van der Waals, cation–π, π–π, ion–ion, dipole–dipole, steric, and hydrophobic interactions with the receptor, with water channels and bridging water molecules mediating hydrogen bonds, as well as metal–ion coordination, as we have recently demonstrated in the latter case (15, 16). Therefore, molecular shape can be considered a “straw-man” alternative to the vibration theory when describing the differing affinities of ligands bound to GPCRs (17, 18), including isotopomers (19, 20). Some of these attractive and repulsive interactions were identified in 1940 by Pauling and Delbrück (21), who note that interacting biomolecules “must have complementary surfaces, like die and coin, and also a complementary distribution of active groups.” In addition, shape-related features are misrepresented by vibration theory proponents. For example, Franco et al. (17) stated: “Given that proteins are chiral, a shape-only theory cannot account for the identical odors of most enantiomeric pairs,” echoing similar comments by Turin (22): “One would therefore generally expect enantiomers to have completely different smells. This is emphatically not the case.” However, these assertions are clearly at odds with the highly developed ability of mice and other mammals to discriminate an array of nonpheromonal chiral odorant enantiomeric pairs (23–25), with the divergent in vitro responses to enantiomers by different combinations of ORs (26) and, in particular, with the highly selective response of the musk-sensitive mouse receptor, MOR215-1, to (R)-muscone (“l-muscone”) compared with (S)-muscone (“d-muscone”) (27).In addition to our concerns regarding shape, a second issue relates to describing how different smells are perceived, that is, the perception of an odorant. It is known that in vivo perception of odorants reflects the totality of perireceptor events as well as odorant–OR interactions (reception). Volatile odorants enter the nasal passage, where they dissolve in the nasal mucus overlying the olfactory epithelium and are then rapidly detected by ORs on the cilia of the olfactory sensory neurons, ultimately leading to signaling (28, 29). It is the mechanism of odorant–OR interactions, the reception of the odorant, that we seek to examine with isotopomers to determine whether the vibration theory is plausible, displaying isotope effects, because perception could be influenced by isotope effects due to the perireceptor events involving mucosal components, such as enzymes, mucopolysaccharides, salts, and antibodies.Whether deuterated and nondeuterated odorant isotopomers can be distinguished by smell and, even if they can, whether this distinction validates the vibration theory is a matter of contention. A 2001 paper by Haffenden et al. (30) reported that benzaldehyde-d₆ gave a statistically significant difference in odor perception relative to normal benzaldehyde, in support of the vibration theory. However, this study has been criticized for lacking double-blind controls to eliminate bias and because it used an anomalous version of the duo-trio test (31). Furthermore, the study failed to account for perireceptor events, namely, the enzyme-mediated conversion of odorants that has been shown to occur in nasal mucus. For example, benzaldehyde is converted to benzoic acid (32), a reaction potentially subject to significant primary isotope effects (2, 33, 34), which could explain the difference in odor perception for the benzaldehyde isotopomers. Earlier claims that human subjects can distinguish odors of acetophenone isotopomers (9, 35) have been shown to be untrue (14, 31). Recent studies indicate that Drosophila melanogaster can distinguish acetophenone isotopomers (36, 37) and that Apis mellifera L., the honey bee, can be trained to discriminate pairs of isotopomers (38). These studies differ from earlier insect studies in which isotopomer discrimination was not found. For example, systematic deuteration of 4-(p-hydroxyphenyl)-2-butanone acetate, a Dacus cucurbitae Coquillett (the male melon fly) attractant, did not affect the attractiveness of the compound to the fly (39); deuteration of alarm pheromones failed to alter the response toward these compounds by Pogonomyrmex badius worker ants (40); and honey bees could not distinguish between deuterated and nondeuterated nitrobenzene (41).Concerns have been raised (42) about aspects of the Drosophila study (36), which is “behavioural and not at the receptor level” (2) (also a concern with the Apis study). Also, given that the ORs and their downstream signaling in Drosophila and humans are completely unrelated, the Drosophila study should not be considered predictive of the ability of humans to distinguish isotopomers (2, 17). In view of the above discussion, it is interesting that in a blinded behavioral study, smell panelists distinguished between deuterated and nondeuterated isotopomers of cyclopentadecanone (1; Fig. 1A) and other musk odorants (14).Open in a separate windowFig. 1.(A) Preparation of deuterated 1–3. Deuterium could be selectively introduced, or selectively removed, adjacent to the carbonyl group using D₂O/K₂CO₃ or H₂O/K₂CO₃, respectively, at 130 °C; global replacement of all hydrogens could be achieved with Rh/C in D₂O at 150 °C. Repetition led to more complete deuteration as well as reduction of 1 to 3 and 2; oxidation of 2 gave 1 with ∼98% deuteration. Chromatography of deuterated 1 with freshly distilled pentane followed by repeated recrystallization from methanol/water to constant melting point gave samples showing no new peaks in their ¹H NMR spectra, other than very weak peaks corresponding to those peaks seen in undeuterated 1. (B) Deuterated (97%) muscone 4 was prepared via alcohol 5 as above. (C) 8-d₅ and 2,4,5,7-tetrathiaoctane-d_10, (9-d₁₀; 98% deuterium) were prepared as shown. Details of these syntheses are provided in SI Appendix.Here, we study the response of human musk-sensitive OR5AN1, identified through screening of heterologously expressed human ORs, to cyclopentadecanone (1) and muscone (4) isotopomers. We also present pharmacological data on the response of mouse ORs to deuterated and nondeuterated acetophenone and benzaldehyde, as well as selected ¹³C isotopomers. In addition, we present related studies on the response of various human and mouse ORs to other deuterated and nondeuterated odorants, including (methylthio)-methanethiol (MTMT, 8; Fig. 1C) and bis(methylthiomethyl) disulfide (9), studied in connection with our investigation of the role of copper coordination in the recognition of both sulfur-containing odorants by the mouse (methylthio)methanethiol receptor, MOR244-3 (15, 16). Insofar as the ability to distinguish odors of isotopomers directly tests the predictions of the vibration theory, the comparative response of human and mouse ORs to isotopomers of these selected ligands in the heterologous OR expression system constitutes a robust test of the vibration theory. Finally, we discuss the basis for recent vibration theories of olfaction and supporting computational evidence (37, 43–47) in light of well-established electron transfer theories (48). We point out that key assumptions underlying the vibration theory lack experimental support and are missing important physical features expected for biological systems.     

A preliminary study on plasma concentration achieved following intrapterygomandibular space injection of dexamethasone as a route of drug delivery with lignocaine inferior alveolar nerve block–correlation of clinical effects

Purpose

To determine the quantity of dexamethasone plasma concentration achieved following intrapterygomandibular space injection of dexamethasone when co-administered with inferior alveolar nerve block correlating with the clinical effects in the postoperative phase.

Objective

A preliminary prospective study to evaluate the dexamethasone plasma concentration achieved following intrapterygomandibular space injection of dexamethasone with 2% lignocaine inferior alveolar nerve block to achieve hemi-mandibular anesthesia for minor oral surgical procedures and derive clinical correlations.

Background

Dexamethasone is a glucocorticoid, chiefly used for the management of postsurgical sequelae like trismus and swelling in maxillofacial surgical practice. Conventionally, parenteral dexamethasone is administered via intravenous or intramuscular route. Intrapterygomandibular space injection is a novel route of steroid delivery described in literature. For minor oral surgical procedures in maxillofacial surgical practice requiring inferior alveolar nerve block, dexamethasone can be administered along with local anesthetic through a single injection as a mixture (twin mix).

Methods

Prospective double-blind randomized clinical trial was designed to evaluative plasma concentration of dexamethasone achieved following injection of a freshly prepared mixture of 1.8 ml of 2% lignocaine with adrenaline (1:200000) and 1 ml (4 mg) dexamethasone [2.8 ml solution of twin mix] in the pterygomandibular space. The 30 candidates included for the trial were randomly split into three study groups (ten each)—(1) control group (C); (2) intramuscular group (IM); (3) intraspace group (IS).

Results

The mean plasma dexamethasone concentration at 30 min postinjection in group IM was 226.41?±?48.67 ng/ml and for IS group it was 209.67?±?88.13 ng/ml. Post hoc (Bonferroni-Holm test) intergroup comparison for plasma dexamethasone concentration (IM and IS) was found statistically insignificant (P?=?0.605).

Conclusion

Intraspace route of drug administration can be utilized to deliver dexamethasonized local anesthetics safely with predictable clinical effects in the patients requiring mandibular minor oral surgery under local anesthesia.

     

Drivers of fatal bird collisions in an urban center

Millions of nocturnally migrating birds die each year from collisions with built structures, especially brightly illuminated buildings and communication towers. Reducing this source of mortality requires knowledge of important behavioral, meteorological, and anthropogenic factors, yet we lack an understanding of the interacting roles of migration, artificial lighting, and weather conditions in causing fatal bird collisions. Using two decades of collision surveys and concurrent weather and migration measures, we model numbers of collisions occurring at a large urban building in Chicago. We find that the magnitude of nocturnal bird migration, building light output, and wind conditions are the most important predictors of fatal collisions. The greatest mortality occurred when the building was brightly lit during large nocturnal migration events and when winds concentrated birds along the Chicago lakeshore. We estimate that halving lighted window area decreases collision counts by 11× in spring and 6× in fall. Bird mortality could be reduced by ∼60% at this site by decreasing lighted window area to minimum levels historically recorded. Our study provides strong support for a relationship between nocturnal migration magnitude and urban bird mortality, mediated by light pollution and local atmospheric conditions. Although our research focuses on a single site, our findings have global implications for reducing or eliminating a critically important cause of bird mortality.

North America has lost nearly one-third of its birdlife in the last half-century, with migratory species experiencing particularly acute declines (1). Fatal collisions with built structures represent a major source of direct, human-caused bird mortality across North America, second only to predation by domestic cats (2). Estimates indicate that between 365 million and 988 million birds die annually in collisions with buildings in the United States, with another 16 million to 42 million annual deaths in Canada (2, 3). Birds may collide with glass windows because they reflect the surrounding environment or allow birds to perceive a seemingly open pathway to the interior of the building (4). For the billions of birds that migrate at night, outdoor lighting (e.g., streetlights and floodlights) and interior lighting from buildings may be disorienting and draw birds into built-up areas, at high risk to collide with infrastructure (5–8). Light pollution not only alters nocturnal migratory behavior on a large scale (5, 7), but is also an acute conservation concern. Nocturnal collisions with well-lit communication towers alone are estimated to kill appreciable percentages of the populations of sensitive species (9).Avian collisions with lighted structures have been documented in the scientific literature as early as the 19th century (10–12). In recent decades, this link between collisions and light pollution has been the subject of detailed investigation (8, 13–16). Observers of bird–building collisions and tower kills have long remarked on the apparent influence of meteorological factors such as cloud ceiling, fog, frontal passage, and abrupt changes in conditions, all of which have been associated with large mortality events (10, 13, 17–24). Steady-burning lights may be particularly hazardous (25). Due to high building density and intensity of artificial lighting, cities are of particular concern. Reports of mass collisions at lighted buildings in urban areas are frequent in both the popular and scientific press (13, 19–21, 26).Understanding, predicting, and preventing collision mortality are areas of active scientific inquiry and priorities for policymakers (1, 13). Collisions occur more frequently during migration seasons and impact numerous species of migratory birds (29), and recent work suggests that nocturnal migratory movements can be useful for predicting bird–window collisions (30). Lights-out programs, which encourage the public to extinguish outdoor lighting to protect migratory birds, are receiving increasing attention (13). The act of extinguishing lighting allows birds to immediately return to normal, safe behavior (7) and reduces mortality at lighted buildings (13). Presently, advisories are generally issued for a given time period (e.g., peak migration periods) or on specific nights when weather conditions are favorable for large migratory movements [e.g., using migration forecasting, (31, 32)].Here, we integrate meteorological, migration-intensity, and window-radiance data to understand how these factors interact to cause bird collisions. We use a 21-y dataset of fatal collisions recorded at a single large building (McCormick Place Lakeside Center) in Chicago, IL (Fig. 1), to understand the behavioral, environmental, and anthropogenic drivers of these mortality events. Chicago poses the greatest potential risk from light pollution to migrating birds of all cities in the United States (33), and over 40,000 dead birds have been recovered from McCormick Place alone since 1978 (Figs. 2 and ​and3).3). Since 2000, we have recorded the number of birds and the lighting status of each window bay during dawn collision monitoring. Nocturnal lighting at McCormick Place correlates positively with bird collisions in many songbird species (34), but this association has not been quantified in the context of other important factors, including migration intensity and weather conditions. We estimate the effect of window lighting on collision counts and assess how the intensity of nocturnal bird migration mediates this relationship. We also test whether wind and weather conditions may magnify these associations. Finally, we investigate the spatiotemporal scales at which weather and migration data best explain collision mortality, identifying the times of night and areas of airspace associated with these events.Open in a separate windowFig. 1.Location of McCormick Place along the Chicago lakefront. The Lakeside Center building monitored in this study is highlighted in red in a three-dimensional rendering.Open in a separate windowFig. 2.Summary of collisions recorded at McCormick Place and regional bird migration between 2000 and 2020. (Upper) Individual years are drawn in different colors. Dates are given for mortality events totaling more than 50 birds. Pie charts show the family (fam.) composition of collected birds, with families representing less than 5% of total collisions merged into a single “other” category. (Lower) Summed annual migration passage at the KLOT radar in estimated number of individual birds (years colored). (Lower, Inset) Summed seasonal passage totals in estimated number of birds crossing a 75-km transect, with each point representing a year. Estimates are based on methods from ref. 35.Open in a separate windowFig. 3.Recorded collisions by year and window lighting. (A) Collisions recorded at McCormick Place between 1982 and 2020 for spring (light gray) and fall (dark gray) seasons. Horizontal lines with numeric labels show average seasonal collision totals before and after the window-lighting regime changed from fully lighted to partially lighted in 1999. The year 1997 is not shown because construction limited access to the site during that year. (B) Mean recorded daily collisions by window-lighting status from 2000 to 2020.     

Miliary tuberculosis with left brachial monoplegia: A case report

Tuberculoma of the brain is a major neurological problem in developing countries accounting for 12 to 30 per cent of all intracranial masses. It often presents with focal neurological symptoms or seizures. Simultaneous occurrence of brain tuberculoma with miliary mottling in the lungs is uncommon in the immunocompetent patient. We report only the second case of monoplegia and miliary tuberculosis, wherein the patient presented with acute onset left brachial monoplegia, upper motor neuron facial palsy, and fever with an MRI of the brain showing multiple granulomas and chest x-ray showing miliary mottling. The patient’s neurological deficit started to resolve with corticosteroids and anti-tubercular treatment.