Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model.

Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the "gold-standard" preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.

Immune durability and protection against SARS-CoV-2 re-infection in Syrian hamsters.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a pandemic. As immunity to endemic human coronaviruses (i.e. NL63 or OC43) wanes leading to re-infection, it was unknown if SARS-CoV-2 immunity would also decline permitting repeat infections. Recent case reports confirm previously infected individuals can become re-infected; however, re-infection may be due to heterogeneity in the initial infection or the host immune response, or may be the result of infection with a variant strain that escapes pre-existing immunity. To control these variables, we utilized the Syrian hamster model to evaluate the duration of immunity and susceptibility to re-infection with SARS-CoV-2. Hamsters were given a primary mock or SARS-CoV-2 infection (culture media or 105 TCID50 USA/WA1/2020 isolate, respectively). Mock and SARS-CoV-2 infected hamsters were then given a secondary SARS-CoV-2 infection at 1, 2, 4, or 6 months post-primary infection (n = 14/time point/group). After the primary SARS-CoV-2 infection, hamsters developed anti-spike protein IgG, IgA, and neutralizing antibodies, and these antibodies were maintained for at least 6 months. Upon secondary SARS-CoV-2 challenge, previously SARS-CoV-2 infected animals were protected from weight loss, while all previously mock-infected animals became infected and lost weight. Importantly, despite having high titres of antibodies, one SARS-CoV-2 infected animal re-challenged at 4 months had a breakthrough infection with replicating virus in the upper and lower respiratory tract. These studies demonstrate immunity to SARS-CoV-2 is maintained for 6 months; however, protection may be incomplete and, even in the presence of high antibody titres, previously infected hosts may become re-infected.

Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed Indirect ELISA: Practical considerations.

AIM: The work was conducted to diagnose peste des petits ruminants (PPR) outbreak through an in house developed indirect ELISA (thereafter referred as iELISA) its comparison with other available diagnostic tests and description of practical considerations in its development, utility and limitations. MATERIALS AND METHODS: An outbreak resembled to PPR occurred in two different places of southern Gujarat viz. Vapi and Navsari, affecting 622 animals, including both goat (n = 476) and sheep (n = 146). Animals displayed the typical signs of PPR at Vapi; however diarrhea was the inconsistent feature in animals of Navsari. The affection caused morbidity of 100% and mortality were 73.68% (n = 392/532) and 56.67% (n = 51/90) in Vapi and Navsari outbreaks, respectively. Relevant ante mortem and post mortem samples were collected from representative animals. At the outset of the epidemic no kit was available with us, so agar gel immunodiffusion (AGID) was carried out and a commercial ELISA (cELISA) kit was ordered for making diagnosis through antibody demonstration. Meanwhile, an iELISA was developed in house using PPR vaccine as antigen and protein G conjugated HRPO antibody as detector. Histopathology and results of sandwich ELISA were also used to diagnose PPR virus (PPRV) in the outbreak. RESULTS: The iELISA developed had detected PPRV antibodies in 22/24 samples (91.66%). Significant difference was observed in disease sensitivity pattern of two species by Chi-square test. While AGID failed to detect antibodies in any sample. Results were reconfirmed by comparing with commercially available cELISA kit. CONCLUSION: PPR is an economically important disease and for the rapid diagnosis of PPR the in house developed antibody capture iELISA can be a suitable cost effective alternative.

In vivo measurement of the surface energy of human fingernail plates.

The surface energy of the human nail plate is expected to influence the adhesion of microorganisms (and subsequent colonization and infections) as well as that of medicines (and subsequent drug permeation) and of cosmetics. The aim of the study was therefore to measure the surface energy of nail plates in vivo. The surface energy of healthy human fingernails (untreated, hydrated and abraded) and of hoof membranes (often used as a model for the nail plate) was estimated from contact angle measurements of liquids (water, formamide, diiodomethane and glycerol) on the nail plate and subsequent computation using the Lifshitz-van der Waals/acid-base (LW-AB) approach. The surface energy of untreated fingernail plates was found to be 34 mJ m(-2) . Most of this total energy was from the apolar Lifshitz-van der Waals component. When the polar component of the surface energy was analysed, the electron donor component was considerably larger than the electron acceptor one. Hydrating the nail plate had no significant influence on the surface energy. In contrast, abrasion caused a small, but statistically significant increase in the apolar surface energy component. The surface energy of bovine hoof membrane was similar to that of the fingernail plate. We conclude that the human fingernail plate is a low-energy surface and that bovine hoof membranes may be used as a substitute for the nail plate in certain experiments.

Stability-indicating Simultaneous HPTLC Method for Olanzapine and Fluoxetine in Combined Tablet Dosage Form.

A rapid, selective and stability-indicating high performance thin layer chromatographic method was developed and validated for the simultaneous estimation of olanzapine and fluoxetine in combined tablet dosage form. Olanzapine and fluoxetine were chromatographed on silica gel 60 F(254) TLC plate using methanol:toluene (4:2 v/v) as the mobile phase and spectrodensitometric scanning-integration was performed at a wavelength of 233 nm using a Camag TLC Scanner III. This system was found to give compact spots for both olanzapine (R(f) value of 0.63+/-0.01) and fluoxetine (R(f) value of 0.31+/-0.01). The polynomial regression data for the calibration plots showed good linear relationship with r(2)=0.9995 in the concentration range of 100-800 ng/spot for olanzapine and 1000-8000 ng/spot for fluoxetine with r(2)=0.9991. The method was validated in terms of linearity, accuracy, precision, recovery and specificity. The limit of detection and the limit of quantification for the olanzapine were found to be 30 and 100 ng/spot, respectively and for fluoxetine 300 and 1000 ng/spot, respectively. Olanzapine and fluoxetine were degraded under acidic, basic and oxidation degradation conditions which showed all the peaks of degraded product were well resolved from the active pharmaceutical ingredient. Both drugs were not further degraded after thermal and photochemical degradation. The method was found to be reproducible and selective for the simultaneous estimation of olanzapine and fluoxetine. As the method could effectively separate the drugs from their degradation products, it can be employed as a stability-indicating method.

Psychosomatic disorders in pediatrics.

Psychosomatic symptoms are by definition clinical symptoms with no underlying organic pathology. Common symptoms seen in pediatric age group include abdominal pain, headaches, chest pain, fatigue, limb pain, back pain, worry about health and difficulty breathing. These, more frequently seen symptoms should be differentiated from somatoform or neurotic disorders seen mainly in adults. The prevalence of psychosomatic complaints in children and adolescents has been reported to be between 10 and 25%. These symptoms are theorized to be a response to stress. Potential sources of stress in children and adolescents include schoolwork, family problems, peer pressure, chronic disease or disability in parents, family moves, psychiatric disorder in parents and poor coping abilities. Characteristics that favour psychosomatic basis for symptoms include vagueness of symptoms, varying intensity, inconsistent nature and pattern of symptoms, presence of multiple symptoms at the same time, chronic course with apparent good health, delay in seeking medical care, and lack of concern on the part of the patient. A thorough medical and psychosocial history and physical examination are the most valuable aspects of diagnostic evaluation. Organic etiology for the symptoms must be ruled out. Appropriate mental health consultation should be considered for further evaluation and treatment.

Calcium-dependent binding of annexin 12 to phospholipid bilayers: stoichiometry and implications.

Annexins (ANXs) are a superfamily of proteins whose functional hallmark is Ca2+-dependent binding to anionic phospholipids. Their core domains are usually composed of a 4-fold repeat of a conserved amino acid sequence, with each repeat containing a type II Ca2+ binding site that is generally thought to mediate Ca2+-dependent binding to the membrane. We now report that ANX12 binding to phospholipid vesicles is highly cooperative with respect to Ca2+ concentration (Hill constant approximately 7), thereby suggesting that more than the four well-characterized type II Ca2+ binding sites are involved in phospholipid binding. Two independent approaches, a novel 45Ca2+ copelleting assay and isothermal titration calorimetry, indicate a stoichiometry of approximately 12 mol of Ca2+/mol of ANX12 for binding to phospholipid vesicles. On the basis of the "low-affinity" Ca2+-binding sites in a number of ANX X-ray crystal structures, we propose a model for ANX12 bilayer binding that involves three types of Ca2+ sites in each of the four repeats. In this model, there is a complementarity between the spacing of the ANX12 Ca2+ binding sites and the spacing of the phospholipid headgroups in bilayers. We tested the implications of the model by manipulating the physical state of vesicles composed of phospholipids with saturated acyl chains with temperature and measuring its influence on ANX12 binding. ANX12 bound to vesicles in a Ca2+-dependent manner when the vesicles were in the liquid crystal phase but not when the phospholipid was in the gel phase. Furthermore, ANX12 bound initially to fluid bilayers remained bound when cooled to 4 degrees C, a temperature that should induce the gel phase transition. Overall, these studies suggest that ANX12 is well suited to being a Ca2+ sensor for rapid all-or-none intercellular membrane-related events.

Recent advances in the hearing assessment of children.

The remarkable specificity and sensitivity of otoacoustic emissions (OAEs) in identifying cochlear dysfunction, and the speed and objectivity with which the test can be conducted has made the OAE procedure the 'standard-of-care' in pediatric audiology assessment. Together with the auditory brainstem responses (ABRs), the OAE procedure not only separates sensory from neural impairment, but also facilitates early audiologic diagnosis and management. This article describes some unique applications of the OAE procedure in the diagnosis, monitoring and treatment of auditory dysfunction.

Contraception in the adolescent: an update.

Contraception remains an important part of national efforts to reduce adolescent pregnancy in the United States. A number of safe and effective contraceptive methods are available for our youth, including abstinence, barrier methods, oral contraceptives, Depo-Provera, and Norplant. Research over the past few decades has resulted in a variety of oral contraceptives with reduced amounts of hormones and reduced side-effects. A number of methods have received approval by the Food and Drug Administration since the last review in 1980, including emergency contraceptives, depomedroxyprogesterone acetate, and the cervical cap. The use of condoms and vaginal spermicides continues to be recommended for all sexually active adolescents to reduce (not eliminate) the risk for acquiring sexually transmitted diseases. A polyurethane condom is now available, in addition to the latex condom and other barrier contraceptives, including the following: diaphragm, cervical cap, vaginal sponge, female condom and vaginal spermicides. Because of continuing concerns about pelvic inflammatory disease related to intrauterine devices, currently available intrauterine devices are not recommended for most adolescents. Abortion is not considered as a contraceptive method.

Treatment of attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a common disorder among children and adolescents with reported prevalence rates of between 3 and 10%. Recent reports suggest that a multimodal treatment approach is preferable to address many symptoms of ADHD and its associated problems for the children, the family, and the school. Stimulant medications remain the mainstay of treatment and are highly effective in more than 75% of patients. Improvement in the core symptoms of inattention, impulsivity, and hyperactivity are most noticeable. Recently approved extended release preparation of methylphenidate will allow once a day dosing. Reports of effectiveness of some non-stimulant medications such as bupropion, especially for adolescents, appear promising. A number of behavioral and psychosocial interventions can be used effectively as part of multimodal approach to address many ADHD-related problems. This article provides an update on practical information on the treatment of children and adolescents with ADHD who do not have other associated psychiatric disorders.

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated stimulation of glutamate and GABA release in the rat striatum in vivo: a dual-label microdialysis study.

The existence of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate autoreceptors on glutamate nerve terminals in vitro has recently been demonstrated using synaptosomal and brain slice preparations. In the present study we have used a modification of a rapid dual-label intracerebral microdialysis method, previously developed by Young and co-workers(80,81) for the study of presynaptic mechanisms of neurotransmitter release, to investigate whether presynaptic AMPA receptors also play a role in the control of striatal glutamate release in vivo. For comparative purposes, the action of locally applied AMPA on striatal GABA release in vivo was also monitored. Local application of AMPA (0.01-100 microM), by reverse dialysis, into the striatum resulted in concentration-dependent increases in the Ca(2+)-dependent efflux of both [3H]L-glutamate and [14C]GABA. Maximum responses reached 142.0+/-6.5% and 166.8+/-7.7% of basal efflux for [3H]L-glutamate and [14C]GABA, respectively. No marked behavioural changes were observed at any dose of the agonist. Unexpectedly, the AMPA-evoked responses were not potentiated by the AMPA receptor desensitization inhibitors cyclothiazide (10-100microM) or aniracetam (1mM). Consistent with this finding, AMPA-stimulated [3H]L-glutamate and [14C]GABA efflux were significantly attenuated by co-perfusion with the selective, competitive AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo(F)quinoxaline-2,3-dione (100microM) but not 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (100microM), a non-competitive AMPA receptor antagonist known to interact with the cyclothiazide site to control AMPA receptor function. The broad spectrum ionotropic glutamate receptor antagonist, kynurenic acid (100-1000microM) also markedly inhibited the AMPA-evoked responses in the striatum in vivo. None of the antagonists, when given alone, influenced basal efflux of [3H]L-glutamate suggesting a lack of tonic regulatory control of glutamate release via presynaptic AMPA-type autoreceptors in the rat striatum. These results demonstrate the presence of presynaptic AMPA receptors, of a novel cyclothiazide- and aniracetam-insensitive subtype, on presynaptic nerve terminals in the rat striatum in vivo, acting to enhance glutamate and GABA release. Our data support the concept of AMPA receptor heterogeneity in vivo, a finding which may facilitate the development of novel, more selective drugs for the treatment of a range of neurological disorders associated with abnormal cerebral glutamate release. The pharmacological profile of these novel presynaptic receptors is currently under investigation.

Pulmonary effects of smoking.

The problems of tobacco addiction have evolved over centuries. The possible relationship between smoking and oral cancer was recognized as early as the 19th century. The use of tobacco results in an estimated 4 million deaths each year worldwide. Approximately 3,000 adolescents start smoking every day; 4.5 million children and adolescents smoke cigarettes; 1 million use smokeless tobacco. This article reviews the effects of environmental tobacco smoke and primary smoking on lung health and maturation and the pathophysiology of smoking-related pulmonary disease. Smoking prevention and timely smoking cessation will significantly reduce the risk of not only lung diseases (chronic obstructive pulmonary disease, cancer, chronic bronchitis, asthma, etc.) but also suboptimal lung growth during preadolescent and adolescent years.

Office interventions for adolescent smokers.

Smoking-prevention efforts can be undertaken at a national level, with enactment and enforcement of laws on the use of tobacco products by youth; at the state and local level, with involvement of community organizations; and through school systems, with education regarding the harmful effects of tobacco use. This review, however, focuses on the role of individual practitioners who also can make significant contributions by working at an individual level to incorporate prevention and treatment strategies in their daily medical practice. This article reviews two types of smoking cessation interventions-behavioral and pharmacologic. Currently available data on the prevention and treatment of nicotine addiction in adolescents, particularly pharmacotherapy, are quite limited. The individual clinician can contribute to prevention and treatment of tobacco use among children and adolescents by using many of the known behavioral and pharmacologic strategies.

Sports injuries in adolescents.

Musculoskeletal injuries are the most common injuries in youth sports. A number of growth-related factors unique to the adolescent age group play an important role in the epidemiology, types, and management of these injuries. These mainly relate to the physical and psychosocial growth and development during adolescence and are discussed in this article. Overuse injuries are more commonly seen in this age group in the primary care setting. Shoulder impingement, elbow and shoulder injuries related to throwing, stress injuries of the wrist, spondylolysis, and anterior knee pain are some of the overuse injuries reviewed in this article.

Sports related mild traumatic brain injury in adolescents.

While head injuries are not common in youth sports, they may have catastrophic results. Concussion is also referred to as mild traumatic brain injury (MTBI). Although the occurrence of head injuries has been studied more extensively in American football, the findings have wider application for any MTBI. Recently, more attention has been directed at other sports in which both boys and girls participate. The diagnosis of MTBI is based on subjective findings and subtle changes in mental status. Other cerebral injuries requiring emergent or urgent neurosurgical attention should be ruled out. Objective tests such as CT scans and Magnetic Resonance Imaging (MRI) are usually without significant findings in MTBI. Neuropsychological testing may demonstrate areas of deficiencies, however, results may be difficult to interpret because of confounding factors. Complications following MTBI have been known to occur. The most catastrophic of these is second impact syndrome. There are a variety of guidelines for return to play following a concussion, which have been designed in an effort to avoid problems such as second impact syndrome. These guidelines are based more on clinical experience than on scientific evidence. Education, good training and coaching techniques, improved equipment, and rule changes and enforcement, can all help in curbing the sports related head injuries in adolescents.

Annexins V and XII insert into bilayers at mildly acidic pH and form ion channels.

The functional hallmark of annexins is the ability to bind to the surface of phospholipid membranes in a reversible, Ca(2+)-dependent manner. We now report that human annexin V and hydra annexin XII reversibly bound to phospholipid vesicles in the absence of Ca(2+) at low pH; half-maximal vesicle association occurred at pH 5.3 and 5. 8, respectively. The following biochemical data support the hypothesis that these annexins insert into bilayers at mildly acidic pH. First, a photoactivatable reagent (3-trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine) which selectively labels proteins exposed to the hydrophobic domain of bilayers reacted with these annexins at pH 5.0 and below but not at neutral pH. Second, in a Triton X-114 partitioning assay, annexins V and XII act as integral membrane proteins at low pH and as hydrophilic proteins at neutral pH; in the presence of phospholipids half-maximal partitioning into detergent occurred at pH approximately 5.0. Finally, annexin V or XII formed single channels in phospholipid bilayers at low pH but not at neutral pH. A model is discussed in which the concentrations of H(+) and Ca(2+) regulate the reversible conversion of three forms of annexins-soluble, peripheral membrane, and transmembrane.

Romance with the automobile in the 20th century: implications for adolescents in a new millennium.

Nearly three-fourths of deaths among American adolescents and young adults result from only four causes: motor vehicle accidents, other unintentional injuries, homicide, and suicide. Thirty percent of those deaths result from motor vehicle accidents, the number one cause of death among adolescents. A number of factors that influence the morbidity and mortality are associated with driving. Compared to other countries, it is easier for American adolescent to obtain a relatively inexpensive license and gain access to a car. For the young driver, adolescent development and increased risk taking, inexperience, dangerous driving behavior, and alcohol-related factors are of special significance. In this article, we review recent crash statistics as well as effectiveness of various preventive measures, including driver education, graduated licensing, alcohol-related measures, and vehicle-related factors. Graduated licensing and alcohol-related measures have been the most effective measures so far.

Sport participation, risk taking, and health risk behaviors.

Adolescents participate in sports for a variety of reasons. Some seem to enjoy participating in what some might consider very-high-risk or "extreme" sports activities. For some adolescents risk taking becomes pervasive and can be detrimental to normal health and development. The majority of adolescents will do well in the context of athletics, and the many positive benefits of regular physical activity and sports participation should be appropriately emphasized. However, a subset of adolescents may be at greater risk for adverse consequences. This article reviews the reasons for participation and attrition from sports, the phenomenon of thrill seeking in sports, certain risk-taking behaviors of athletes, and studies comparing health risk behaviors in athletes and non-athletes.

Annexin 5 mediates a peroxide-induced Ca(2+) influx in B cells.

Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.