Carotid body dysregulation contributes to Long COVID symptoms.

BACKGROUND: The symptoms of long COVID, which include fatigue, breathlessness, dysregulated breathing, and exercise intolerance, have unknown mechanisms. These symptoms are also observed in heart failure and are partially driven by increased sensitivity of the carotid chemoreflex. As the carotid body has an abundance of ACE2 (the cell entry mechanism for SARS-CoV-2), we investigated whether carotid chemoreflex sensitivity was elevated in participants with long COVID. METHODS: Non-hositalised participants with long-COVID (n = 14) and controls (n = 14) completed hypoxic ventilatory response (HVR; the measure of carotid chemoreflex sensitivity) and cardiopulmonary exercise tests. Parametric and normally distributed data were compared using Student's unpaired t-tests or ANOVA. Nonparametric equivalents were used where relevant. Peason's correlation coefficient was used to examine relationships between variables. RESULTS: During cardiopulmonary exercise testing the VE/VCO2 slope (a measure of breathing efficiency) was higher in the long COVID group (37.8 ± 4.4) compared to controls (27.7 ± 4.8, P = 0.0003), indicating excessive hyperventilation. The HVR was increased in long COVID participants (-0.44 ± 0.23 l/min/ SpO2%, R2 = 0.77 ± 0.20) compared to controls (-0.17 ± 0.13 l/min/SpO2%, R2 = 0.54 ± 0.38, P = 0.0007). The HVR correlated with the VE/VCO2 slope (r = -0.53, P = 0.0036), suggesting that excessive hyperventilation may be related to carotid body hypersensitivity. CONCLUSIONS: The carotid chemoreflex is sensitised in long COVID and may explain dysregulated breathing and exercise intolerance in these participants. Tempering carotid body excitability may be a viable treatment option for long COVID patients.

Patients with long COVID suffer from breathlessness during exercise, leading to exercise intolerance. We know that SARS-CoV-2, the virus that causes COVID-19, can infect carotid bodies which is a small sensory organ that sends signals to the brain for regulating breathing and blood pressure. This is called the carotid chemoreflex. However, it is not clear if SARS-CoV-2 infection affects carotid chemoreflex. Here, we examine whether the normal functioning of carotid chemoreflex is disrupted in non-hospitalised patients with long COVID and if this is linked to excessive breathing during exercise. Our study shows that carotid chemoreflex is more sensitive in long COVID patients, who are otherwise healthy. The carotid bodies could be a good therapeutic target for treating breathlessness in patients with long COVID.

Impact of the COVID-19 related border restrictions on influenza and other common respiratory viral infections in New Zealand.

BACKGROUND: New Zealand's (NZ) complete absence of community transmission of influenza and respiratory syncytial virus (RSV) after May 2020, likely due to COVID-19 elimination measures, provided a rare opportunity to assess the impact of border restrictions on common respiratory viral infections over the ensuing 2 years. METHODS: We collected the data from multiple surveillance systems, including hospital-based severe acute respiratory infection surveillance, SHIVERS-II, -III and -IV community cohorts for acute respiratory infection (ARI) surveillance, HealthStat sentinel general practice (GP) based influenza-like illness surveillance and SHIVERS-V sentinel GP-based ARI surveillance, SHIVERS-V traveller ARI surveillance and laboratory-based surveillance. We described the data on influenza, RSV and other respiratory viral infections in NZ before, during and after various stages of the COVID related border restrictions. RESULTS: We observed that border closure to most people, and mandatory government-managed isolation and quarantine on arrival for those allowed to enter, appeared to be effective in keeping influenza and RSV infections out of the NZ community. Border restrictions did not affect community transmission of other respiratory viruses such as rhinovirus and parainfluenza virus type-1. Partial border relaxations through quarantine-free travel with Australia and other countries were quickly followed by importation of RSV in 2021 and influenza in 2022. CONCLUSION: Our findings inform future pandemic preparedness and strategies to model and manage the impact of influenza and other respiratory viral threats.

Early transcriptional responses to human enteric fever challenge.

Enteric fever, caused by oral infection with typhoidal Salmonella serovars, presents as a non-specific febrile illness preceded by an incubation period of 5 days or more. The enteric fever human challenge model provides a unique opportunity to investigate the innate immune response during this incubation period, and how this response is altered by vaccination with the Vi polysaccharide or conjugate vaccine. We find that on the same day as ingestion of typhoidal Salmonella, there is already evidence of an immune response, with 199 genes upregulated in the peripheral blood transcriptome 12 hours post-challenge (false discovery rate <0.05). Gene sets relating to neutrophils, monocytes, and innate immunity were over-represented (false discovery rate <0.05). Estimating cell proportions from gene expression data suggested a possible increase in activated monocytes 12 hours post-challenge (P = 0.036, paired Wilcoxon signed-rank test). Furthermore, plasma TNF-α rose following exposure (P = 0.011, paired Wilcoxon signed-rank test). There were no significant differences in gene expression (false discovery rate <0.05) in the 12 hours response between those who did and did not subsequently develop clinical or blood culture confirmed enteric fever or between vaccination groups. Together, these results demonstrate early perturbation of the peripheral blood transcriptome after enteric fever challenge and provide initial insight into early mechanisms of protection.

Aging in females is associated with changes in respiratory modulation of sympathetic nerve activity and blood pressure.

Sympathetic nerve activity (SNA) is tightly coupled with the respiratory cycle. In healthy human males, respiratory modulation of SNA does not change with age. However, it is unclear how this modulation is affected by age in females. We investigated whether respiratory sympathetic modulation is altered in healthy postmenopausal (PMF) versus premenopausal female (YF), and younger male (YM) adults, and determined its relationship to resting blood pressure. Muscle SNA (MSNA; microneurography), respiration (transducer belt), ECG, and continuous blood pressure were measured in 12 YF, 13 PMF, and 12 YM healthy volunteers. Respiratory modulation of MSNA was quantified during two phases of the respiratory cycle: mid-late expiration and inspiration/postinspiration. All groups showed respiratory modulation of MSNA (P < 0.0005). There was an interaction between the respiratory phase and group for MSNA [bursts/100 heartbeats (HB) (P = 0.004) and bursts/min (P = 0.029)], with smaller reductions in MSNA during inspiration observed in PMF versus the other groups. Respiratory modulation of blood pressure was also reduced in PMF versus YF (6 [2] vs. 12 [9] mmHg, P = 0.008) and YM (13 [13] mmHg, P = 0.001, median [interquartile range]). The magnitude of respiratory sympathetic modulation was related to resting blood pressure in PMF only, such that individuals with less modulation had greater resting blood pressure. The data indicate that aging in postmenopausal females is associated with less inspiratory inhibition of MSNA. This correlated with a higher resting blood pressure in PMF only. Thus, the reduced modulation of MSNA could contribute to the age-related rise in blood pressure that occurs in females.NEW & NOTEWORTHY The current study demonstrates that respiratory modulation of sympathetic nerve activity (SNA) is reduced in healthy postmenopausal (PMF) versus premenopausal females (YF). Furthermore, respiratory sympathetic modulation was negatively related to resting blood pressure in postmenopausal females, such that blood pressure was greater in individual with less modulation. Reduced respiratory sympathetic modulation may have implications for the autonomic control of blood pressure in aging postmenopausal females, by contributing to age-related sympathetic activation and reducing acute, respiratory-linked blood pressure variation.

Bruxism, parafunctional oral habits and oral motor problems in children with spastic cerebral palsy: A cross-sectional study.

BACKGROUND: Individuals with spastic cerebral palsy are more predisposed to parafunctional oral activities and oral motor problems because of spasticity. OBJECTIVES: The aim of the study was to evaluate the relationship between the gross motor function classification system score (GMFCS), age, bruxism, parafunctional oral habits and oral motor problems in children with cerebral palsy. METHODS: This cross-sectional study included 63 children with spastic cerebral palsy, aged 3-18 years, with developmental disabilities. The relationship between parentally reported bruxism, parafunctional oral activity rates, oral motor problems, and GMFCS was analysed. RESULTS: The prevalence of bruxism was 52.4%, and the rate decreased as age increased. There was a greater likelihood of bruxism in individuals with tongue thrust (OR [95% CI] = 8.15 [1.4-47.3]) and swallowing problems (OR [95% CI] = 5.78 [1.3-24.68]). CONCLUSION: In children with spastic cerebral palsy, bruxism and the rate of parafunctional oral habits were high, thus affecting oral motor activities. A relationship was found between oral motor problems and increased GMFCS levels, but no relationship was found between bruxism and GMFCS levels. Children with spastic cerebral palsy who display tongue thrust or swallowing problems have an increased likelihood of presenting with bruxism.

Inborn Errors of Immunity in Children With Invasive Pneumococcal Disease: A Multicenter Prospective Study.

BACKGROUND: In settings with universal conjugate pneumococcal vaccination, invasive pneumococcal disease (IPD) can be a marker of an underlying inborn error of immunity. The aim of this study was to determine the prevalence and characterize the types of immunodeficiencies in children presenting with IPD. METHODS: Multicenter prospective audit following the introduction of routinely recommended immunological screening in children presenting with IPD. The minimum immunological evaluation comprised a full blood examination and film, serum immunoglobulins (IgG, IgA and IgM), complement levels and function. Included participants were children in whom Streptococcus pneumoniae was isolated from a normally sterile site (cerebrospinal fluid, pleura, peritoneum and synovium). If isolated from blood, features of sepsis needed to be present. Children with predisposing factors for IPD (nephrotic syndrome, anatomical defect or malignancy) were excluded. RESULTS: Overall, there were 379 episodes of IPD of which 313 (83%) were eligible for inclusion and 143/313 (46%) had an immunologic evaluation. Of these, 17/143 (12%) were diagnosed with a clinically significant abnormality: hypogammaglobulinemia (n = 4), IgA deficiency (n = 3), common variable immunodeficiency (n = 2), asplenia (n = 2), specific antibody deficiency (n = 2), incontinentia pigmenti with immunologic dysfunction (n = 1), alternative complement deficiency (n = 1), complement factor H deficiency (n = 1) and congenital disorder of glycosylation (n = 1). The number needed to investigate to identify 1 child presenting with IPD with an immunologic abnormality was 7 for children under 2 years and 9 for those 2 years old and over. CONCLUSIONS: This study supports the routine immune evaluation of children presenting with IPD of any age, with consideration of referral to a pediatric immunologist.

A year in review: brain barriers and brain fluids research in 2022.

This aim of this editorial is to highlight progress made in brain barrier and brain fluid research in 2022. It covers studies on the blood-brain, blood-retina and blood-CSF barriers (choroid plexus and meninges), signaling within the neurovascular unit and elements of the brain fluid systems. It further discusses how brain barriers and brain fluid systems are impacted in CNS diseases, their role in disease progression and progress being made in treating such diseases.

2022 AAFP/ISFM Cat Friendly Veterinary Interaction Guidelines: Approach and Handling Techniques.

PRACTICAL RELEVANCE: The '2022 AAFP/ISFM Cat Friendly Veterinary Interaction Guidelines: Approach and Handling Techniques' (hereafter the 'Cat Friendly Veterinary Interaction Guidelines') support veterinary professionals with feline interactions and handling to reduce the impact of fear and other protective (negative) emotions, in so doing enhancing feline welfare and In implementing these Guidelines, team satisfaction and cat caregiver confidence in the veterinary team will increase as the result of efficient examinations, better experience, more reliable diagnostic testing and improved feline wellbeing. Veterinary professionals will learn the importance of understanding and appropriately responding to the current emotional state of the cat and tailoring each visit to the individual. CLINICAL CHALLENGES: Cats have evolved with emotions and behaviors that are necessary for their survival as both a predator and prey species. A clinical setting and the required examinations and procedures to meet their physical health needs can result in behavioral responses to protective emotions. Cat friendly interactions require understanding, interpreting and appropriately responding to cats' emotional states and giving them a perceived sense of control while performing the required assessment. EVIDENCE BASE: These Guidelines have been created by a Task Force of experts convened by the American Association of Feline Practitioners and the International Society of Feline Medicine, based on an extensive literature review and, where evidence is lacking, the authors' experience. ENDORSEMENTS: These Guidelines have been endorsed by a number of groups and organizations, as detailed on page 1127 and at catvets.com/interactions and icatcare.org/cat-friendly-guidelines.

Advances in brain barriers and brain fluids research in 2021: great progress in a time of adversity.

This editorial highlights advances in brain barrier and brain fluid research in 2021. It covers research on components of the blood-brain barrier, neurovascular unit and brain fluid systems; how brain barriers and brain fluid systems are impacted by neurological disorders and their role in disease progression; and advances in strategies for treating such disorders.

Covalent Labeling-Mass Spectrometry Provides a Molecular Understanding of Noncovalent Polymer-Protein Complexation.

The delivery of functional proteins to the intracellular space offers tremendous advantages for the development of new therapeutics but is limited by the passage of these large polar biomacromolecules through the cell membrane. Noncovalent polymer-protein binding that is driven by strong carrier-cargo interactions, including electrostatics and hydrophobicity, has previously been explored in the context of delivery of functional proteins. Appropriately designed polymer-based carriers can take advantage of the heterogeneous surface of protein cargoes, where multiple types of physical binding interactions with polymers can occur. Traditional methods of assessing polymer-protein binding, including dynamic light scattering, circular dichroism spectroscopy, and fluorescence-based assays, are useful in the study of new polymer-based carriers but face a number of limitations. We implement for the first time the method of covalent labeling-mass spectrometry (CL-MS) to probe intermolecular surface interactions within noncovalent polymer-protein complexes. We demonstrate the utility of CL-MS for establishing binding of an amphiphilic block copolymer to negatively charged and hydrophobic surface patches of a model protein, superfolder green fluorescent protein (sfGFP), using diethylpyrocarbonate as a pseudo-specific labeling reagent. In addition, we utilize this method to explore differences at the intermolecular surface as the ratio of polymer to protein increases, particularly in the context of defining effective protein delivery regimes. By promoting an understanding of the intermolecular interactions in polymer-protein binding and identifying sites where polymers bind to protein surfaces, noncovalent polymer carriers can be more effectively designed for protein delivery applications.

Protein Binding and Release by Polymeric Cell-Penetrating Peptide Mimics.

There is significant potential in exploiting antibody specificity to develop new therapeutic treatments. However, intracellular protein delivery is a paramount challenge because of the difficulty in transporting large, polar molecules across cell membranes. Cell-penetrating peptide mimics (CPPMs) are synthetic polymers that are versatile materials for intracellular delivery of biological molecules, including nucleic acids and proteins, with superior performance compared to their natural counterparts and commercially available peptide-based reagents. Studies have demonstrated that noncovalent complexation with these synthetic carriers is necessary for the delivery of proteins, but the fundamental interactions dominating CPPM-protein complexation are not well understood. Beyond these interactions, the mechanism of release for many noncovalent carriers is not well established. Herein, interactions expected to be critical in CPPM-protein binding and unbinding were explored, including hydrogen bonding, electrostatics, and hydrophobic interactions. Despite the guanidinium-rich functionality of these polymeric carriers, hydrogen bonding was shown not to be a dominant interaction in CPPM-protein binding. Fluorescence quenching assays were used to decouple the effect of electrostatic and hydrophobic interactions between amphiphilic CPPMs and proteins. Furthermore, by conducting competition assays with other proteins, unbinding of protein cargoes from CPPM-protein complexes was demonstrated and provided insight into mechanisms of protein release. This work offers understanding toward the role of carrier and cargo binding and unbinding in intracellular outcomes. In turn, an improved fundamental understanding of noncovalent polymer-protein complexation will enable more effective methods for intracellular protein delivery.

The NF-κB Nucleolar Stress Response Pathway.

The nuclear organelle, the nucleolus, plays a critical role in stress response and the regulation of cellular homeostasis. P53 as a downstream effector of nucleolar stress is well defined. However, new data suggests that NF-κB also acts downstream of nucleolar stress to regulate cell growth and death. In this review, we will provide insight into the NF-κB nucleolar stress response pathway. We will discuss apoptosis mediated by nucleolar sequestration of RelA and new data demonstrating a role for p62 (sequestosome (SQSTM1)) in this process. We will also discuss activation of NF-κB signalling by degradation of the RNA polymerase I (PolI) complex component, transcription initiation factor-IA (TIF-IA (RRN3)), and contexts where TIF-IA-NF-κB signalling may be important. Finally, we will discuss how this pathway is targeted by aspirin to mediate apoptosis of colon cancer cells.

A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity.

Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence, and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small-molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.

The role of Kingella kingae in pre-school aged children with bone and joint infections.

OBJECTIVES: The Pre-school Osteoarticular Infection (POI) study aimed to describe the burden of disease, epidemiology, microbiology and treatment of acute osteoarticular infections (OAI) and the role of Kingella kingae in these infections. METHODS: Information about children 3-60 months of age who were hospitalized with an OAI to 11 different hospitals across Australia and New Zealand between January 2012 and December 2016 was collected retrospectively. RESULTS: A total of 907 cases (73%) were included. Blood cultures grew a likely pathogen in only 18% (140/781). The peak age of presentation was 12 to 24 months (466/907, 51%) and Kingella kingae was the most frequently detected microorganism in this age group (60/466, 13%). In the majority of cases, no microorganism was detected (517/907, 57%). Addition of PCR to culture increased detection rates of K. kingae. However, PCR was performed infrequently (63/907, 7%). CONCLUSIONS: This large multi-national study highlights the need for more widespread use of molecular diagnostic techniques for accurate microbiological diagnosis of OAI in pre-school aged children. The data from this study supports the hypothesis that a substantial proportion of pre-school aged children with OAI and no organism identified may in fact have undiagnosed K. kingae infection. Improved detection of Kingella cases is likely to reduce the average length of antimicrobial treatment.

Aneurysm severity is suppressed by deletion of CCN4.

Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmaceutical treatment to reverse aneurysm progression could prevent the need for surgery and save both lives and healthcare resources. Since CCN4 regulates cell migration, proliferation and apoptosis, processes involved in aneurysm progression, it is a potential regulator of aneurysm progression. We investigated the role of CCN4 in a mouse aneurysm model, using apolipoprotein-E knockout (ApoE-/-) mice fed high fat diet and infused with Angiotensin II (AngII). Blood pressure was similarly elevated in CCN4-/-ApoE-/- mice and CCN4+/+ApoE-/- mice (controls) in response to AngII infusion. Deletion of CCN4 significantly reduced the number of ruptured aortae, both thoracic and abdominal aortic area, and aneurysm grade score, compared to controls. Additionally, the frequency of vessel wall remodelling and the number of elastic lamina breaks was significantly suppressed in CCN4-/-ApoE-/- mice compared to controls. Immunohistochemistry revealed a significantly lower proportion of macrophages, while the proportion of smooth muscle cells was not affected by the deletion of CCN4. There was also a reduction in both proliferation and apoptosis in CCN4-/-ApoE-/- mice compared to controls. In vitro studies showed that CCN4 significantly increased monocyte adhesion beyond that seen with TNFα and stimulated macrophage migration by more than threefold. In summary, absence of CCN4 reduced aneurysm severity and improved aortic integrity, which may be the result of reduced macrophage infiltration and cell apoptosis. Inhibition of CCN4 could offer a potential therapeutic approach for the treatment of aneurysms.

2021 AAFP Feline Senior Care Guidelines.

The '2021 AAFP Feline Senior Care Guidelines' are authored by a Task Force of experts in feline clinical medicine and serve as an update and extension of those published in 2009. They emphasize the individual patient evaluation and the process of aging, with references to other feline practice guidelines for a more complete discussion of specific diseases. Focusing on each cat encourages and empowers the owner to become a part of the cat's care every step of the way. A comprehensive discussion during the physical examination and history taking allows for tailoring the approach to both the cat and the family involved in the care. Videos and analysis of serial historical measurements are brought into the assessment of each patient. These Guidelines introduce the emerging concept of frailty, with a description and methods of its incorporation into the senior cat assessment. Minimum database diagnostics are discussed, along with recommendations for additional investigative considerations. For example, blood pressure assessment is included as a minimum diagnostic procedure in both apparently healthy and ill cats. Cats age at a much faster rate than humans, so practical timelines for testing frequency are included and suggest an increased frequency of diagnostics with advancing age. The importance of nutrition, as well as senior cat nutritional needs and deficiencies, is considered. Pain is highlighted as its own syndrome, with an emphasis on consideration in every senior cat. The Task Force discusses anesthesia, along with strategies to allow aging cats to be safely anesthetized well into their senior years. The medical concept of quality of life is addressed with the latest information available in veterinary medicine. This includes end of life considerations like palliative and hospice care, as well as recommendations on the establishment of 'budgets of care', which greatly influence what can be done for the individual cat. Acknowledgement is given that each cat owner will be different in this regard; and establishing what is reasonable and practical for the individual owner is important. A discussion on euthanasia offers some recommendations to help the owner make a decision that reflects the best interests of the individual cat.

Non-Covalent Carrier Hydrophobicity as a Universal Predictor of Intracellular Protein Activity.

Over the past decade, extensive optimization of polymeric cell-penetrating peptide (CPP) mimics (CPPMs) by our group has generated a substantial library of broadly effective carriers which circumvent the need for covalent conjugation often required by CPPs. In this study, design rules learned from CPPM development were applied to reverse-engineer the first library of simple amphiphilic block copolypeptides for non-covalent protein delivery, namely, poly(alanine-block-arginine), poly(phenylalanine-block-arginine), and poly(tryptophan-block-arginine). This new CPP library was screened for enhanced green fluorescent protein and Cre recombinase delivery alongside a library of CPPMs featuring equivalent side-chain configurations. Due to the added hydrophobicity imparted by the polymer backbone as compared to the polypeptide backbone, side-chain functionality was not a universal predictor of carrier performance. Rather, overall carrier hydrophobicity predicted the top performers for both internalization and activity of protein cargoes, regardless of backbone identity. Furthermore, comparison of protein uptake and function revealed carriers which facilitated high gene recombination despite remarkably low Cre internalization, leading us to formalize the concept of intracellular availability (IA) of the delivered cargo. IA, a measure of cargo activity per quantity of cargo internalized, provides valuable insight into the physical relationship between cellular internalization and bioavailability, which can be affected by bottlenecks such as endosomal escape and cargo release. Importantly, carriers with maximal IA existed within a narrow hydrophobicity window, more hydrophilic than those exhibiting maximal cargo uptake. Hydrophobicity may be used as a scaffold-independent predictor of protein uptake, function, and IA, enabling identification of new, effective carriers which would be overlooked by uptake-based screening methods.