Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2.

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100 nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12', a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12' N-lobe does not interact with the IQ motif. The CaM12' C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.

In vitro Inhibition of respiratory pathogens by lactobacillus and alpha haemolytic streptococci from Aboriginal and Torres Strait Islander children.

AIMS: To explore the in vitro ability of alpha haemolytic streptococcus (AHS) and lactobacilli (LBs), from Indigenous Australian children, to inhibit the growth of respiratory pathogens (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), also from Indigenous Australian children. METHODS AND RESULTS: The bacterial interference of 91 isolates, from Indigenous Australian children both with and without otitis media (OM) or rhinorrhoea, was investigated using agar overlay and cell-free supernatant. Promising isolates underwent whole genome sequencing to investigate upper respiratory tract tropism, antibiotic resistance and virulence. Antibiotic susceptibility was examined for ampicillin, amoxicillin +clavulanic acid and azithromycin. Differences in the strength of bacterial inferences in relation to OM was examined using a case series of three healthy and three children with OM. LBs readily inhibited the growth of pathogens. AHS were less effective, although several isolates inhibited S. pneumoniae. One L. rhamnosus had genes coding for pili to adhere to epithelial cells. We detected antibiotic resistance genes coding for antibiotic efflux pump and ribosomal protection protein. LBs were susceptible to antimicrobials in vitro. Screening for virulence detected genes encoding for two putative capsule proteins. Healthy children had AHS and LB that were more potent inhibitors of respiratory pathogens in vitro than children with OM. CONCLUSIONS: L. rhamnosus from remote Indigenous Australian children are potent inhibitors of respiratory pathogens in vitro. SIGNIFICANCE AND IMPACT OF STUDY: Respiratory/ear disease are endemic in Indigenous Australians. There is an urgent call for more effective treatment/prevention; beneficial microbes have not been explored. L. rhamnosus investigated in this study are potent inhibitors of respiratory pathogens in vitro and require further investigation.

α-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the α1 Subunit.

The voltage-gated L-type Ca2+ channel CaV1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of CaV1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of CaV1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal CaV1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous CaV1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to CaV1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α11.2 subunit of CaV1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant CaV1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α11.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity.

Predicting functional communication ability in children with cerebral palsy at school entry.

AIM: To explore the value of demographic, environmental, and early clinical characteristics in predicting functional communication in children with cerebral palsy (CP) at school entry. METHOD: Data are from an Australian prospective longitudinal study of children with CP. Children assessed at 18 to 24 and 48 to 60 months corrected age were included in the study. Functional communication was classified at 48 to 60 months using the Communication Function Classification System (CFCS). Predictive variables included communication skills at 18 to 24 months, evaluated using the Communication and Symbolic Behavioural Scales Developmental Profile (CSBS-DP) Infant-Toddler Checklist. Early Gross Motor Function Classification System (GMFCS), Manual Ability Classification System, and motor type and distribution were evaluated by two physiotherapists. Demographic and comorbid variables were obtained through parent interview with a paediatrician or rehabilitation specialist. RESULTS: A total of 114 children (76 males, 38 females) were included in the study. At 18 to 24 months the mean CSBS-DP was 84.9 (SD 19.0). The CFCS distribution at 48 to 60 months was I=36(32%), II=25(22%), III=20(18%), IV=19(17%), and V=14(12%). In multivariable regression analysis, only CSBS-DP (p<0.01) and GMFCS (p<0.01) at 18 to 24 months were predictors of functional communication at school entry. INTERPRETATION: Body structure and function and not environmental factors impact functional communication at school entry in children with CP. This provides valuable guidance for early screening, parent education, and future planning of intervention programs to improve functional communication.

School readiness of children with cerebral palsy.

AIM: To examine school readiness in preschool-age children with cerebral palsy (CP) on three of five domains compared with reported norms of children with typical development (CTD). METHOD: A representative population of 151 preschool-age children with CP (87 males, 64 females; 131 [87%] with spasticity, 17 [11%] dyskinesia, 3 [4%] hypotonia) were assessed at 48 or 60 months corrected age. Children were functioning in the following Gross Motor Function Classification System (GMFCS) levels: I, 74 (49%); II, 17 (11%); III, 14 (9%); IV, 26 (17%); V, 20 (13%). Children's motor performance, self-care, and social function were assessed using the Pediatric Evaluation of Disability Inventory (PEDI) and communication using the Communication and Symbolic Behaviour Scales Developmental Profile (CSBS-DP). Results were compared with a reference sample of CTD (PEDI CTD n=412; CSBS-DP CTD n=790). Linear regression was used to compare these data by functional severity. RESULTS: Children with CP had significantly lower PEDI scores in all domains than CTD. Self-care scores ranged from 0.5 to more than 4SD below CTD, motor performance was 2 to >4SD below CTD, and social function between 0.5 and >4SD below CTD. Fifty-five per cent of children demonstrated significantly delayed communication skills. Non-ambulant children displayed significantly lower scores than ambulant children. INTERPRETATION: Preschool-age children with CP perform significantly below their peers in three of five key readiness-to-learn skill areas including mobility, self-care, social function, and communication abilities. Broader emphasis needs to be placed on multimodal screening and intervention to prepare children with CP for school entry.

Relationship between communication skills and gross motor function in preschool-aged children with cerebral palsy.

OBJECTIVES: To explore the communication skills of children with cerebral palsy (CP) at 24 months' corrected age with reference to typically developing children, and to determine the relationship between communication ability, gross motor function, and other comorbidities associated with CP. DESIGN: Prospective, cross-sectional, population-based cohort study. SETTING: General community. PARTICIPANTS: Children with CP (N=124; mean age, 24mo; functional severity on Gross Motor Function Classification System [GMFCS]: I=47, II=14, III=22, IV=19, V=22). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Parents reported communication skills on the Communication and Symbolic Behavior Scales Developmental Profile (CSBS-DP) Infant-Toddler Checklist. Two independent physiotherapists classified motor type, distribution, and GMFCS. Data on comorbidities were obtained from parent interviews and medical records. RESULTS: Children with mild CP (GMFCS I/II) had mean CSBS-DP scores that were 0.5 to 0.6 SD below the mean for typically developing peers, while those with moderate-severe impairment (GMFCS III-V) were 1.4 to 2.6 SD below the mean. GMFCS was significantly associated with performance on the CSBS-DP (F=18.55, P<.001), with gross motor ability accounting for 38% of the variation in communication. Poorer communication was strongly associated with gross motor function and full-term birth. CONCLUSIONS: Preschool-aged children with CP, with more severe gross motor impairment, showed delayed communication, while children with mild motor impairment were less vulnerable. Term-born children had significantly poorer communication than those born prematurely. Because a portion of each gross motor functional severity level is at risk, this study reinforces the need for early monitoring of communication development for all children with CP.

The Power of Advocacy: Advancing Vision for Everyone to Meet the Sustainable Development Goals.

Advocacy is instrumental to achieving significant policy change for vision. Global advocacy efforts over the past decades enabled recognition of vision as a major public health, human rights, and development issue. The United Nations General Assembly adopted its first-ever Resolution on vision: "Vision for Everyone-Accelerating Action to Achieve the Sustainable Development Goals (SDGs)" on 23 July 2021. The Resolution sets the target and commits the international community to improve vision for the 1.1 billion people living with preventable vision impairment by 2030. To fulfill their commitments, governments and international institutions must act now. Advocacy remains instrumental to mobilize funding and empower governments and stakeholders to include eye health in their implementation agenda. In this paper, we discuss the pivotal role advocacy plays in advancing vision for everyone now and in the post-COVID-19 era. We explore the link between improved eye health and the advancement of SDGs and define the framework and key pillars of advocacy to scaling-up success by 2030.

Upper Respiratory Tract Microbiome of Australian Aboriginal and Torres Strait Islander Children in Ear and Nose Health and Disease.

The objective of this study was to examine the nasal microbiota in relation to otitis media (OM) status and nose health in Indigenous Australian children. Children 2 to 7 years of age were recruited from two northern Australian (Queensland) communities. Clinical histories were obtained through parent interviews and reviews of the medical records. Nasal cavity swab samples were obtained, and the children's ears, nose, and throat were examined. DNA was extracted and analyzed by 16S rRNA amplicon next-generation sequencing of the V3/V4 region, in combination with previously generated culture data. A total of 103 children were recruited (mean age, 4.7 years); 17 (16.8%) were healthy, i.e., normal examination results and no history of OM. The nasal microbiota differed significantly in relation to OM status and nose health. Children with historical OM had greater relative abundance of Moraxella, compared to healthy children, despite both having healthy ears at the time of swabbing. Children with healthy noses had greater relative abundance of Staphylococcus aureus, compared to those with rhinorrhea. Dolosigranulum was correlated with Corynebacterium in healthy children. Haemophilus and Streptococcus were correlated across phenotypes. Ornithobacterium was absent or was present with low relative abundance in healthy children and clustered around otopathogens. It correlated with Helcococcus and Dichelobacter. Dolosigranulum and Corynebacterium form a synergism that promotes upper respiratory tract (URT)/ear health in Indigenous Australian children. Ornithobacterium likely represents "Candidatus Ornithobacterium hominis" and in this population is correlated with a novel bacterium that appears to be related to poor URT/ear health. IMPORTANCE Recurring and chronic infections of the ear (OM) are disproportionately prevalent in disadvantaged communities across the globe and, in particular, within Indigenous communities. Despite numerous intervention strategies, OM persists as a major health issue and is the leading cause of preventable hearing loss. In disadvantaged communities, this hearing loss is associated with negative educational and social development outcomes, and consequently, poorer employment prospects and increased contact with the justice system in adulthood. Thus, a better understanding of the microbial ecology is needed in order to identify new targets to treat, as well as to prevent the infections. This study used a powerful combination of 16S rRNA gene sequencing and extended culturomics to show that Dolosigranulum pigrum, a bacterium previously identified as a candidate protective species, may require cocolonization with Corynebacterium pseudodiphtheriticum in order to prevent OM. Additionally, emerging and potentially novel pathogens and bacteria were identified.

Upper Respiratory Microbiota in Relation to Ear and Nose Health Among Australian Aboriginal and Torres Strait Islander Children.

BACKGROUND: We explored the nasal microbiota in Indigenous Australian children in relation to ear and nasal health. METHODS: In total, 103 Indigenous Australian children aged 2-7 years (mean 4.7 years) were recruited from 2 Queensland communities. Children's ears, nose, and throats were examined and upper respiratory tract (URT) swabs collected. Clinical histories were obtained from parents/medical records. URT microbiota were characterized using culturomics with Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification. Real-time PCR was used to quantify otopathogen (Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis) loads and detect respiratory viruses. Data were analyzed using beta diversity measures, regression modeling, and a correlation network analysis. RESULTS: Children with historical/current otitis media (OM) or URT infection (URTI) had higher nasal otopathogen detection and loads and rhinovirus detection compared with healthy children (all P < .04). Children with purulent rhinorrhea had higher nasal otopathogen detection and loads and rhinovirus detection (P < .04) compared with healthy children. High otopathogen loads were correlated in children with historical/current OM or URTI, whereas Corynebacterium pseudodiphtheriticum and Dolosigranulum pigrum were correlated in healthy children. CONCLUSIONS: Corynebacterium pseudodiphtheriticum and D. pigrum are associated with URT and ear health. The importance of the main otopathogens in URT disease/OM was confirmed, and their role relates to co-colonization and high otopathogens loads.

An end to unsafe care.

Only accountability in the C-suite will bring an end to patient safety failures.

Probiotics in the treatment of otitis media. The past, the present and the future.

Otitis media (OM) is one of the most common infectious diseases in children and the leading cause for medical consultations and antibiotic prescription in this population. The burden of disease associated with OM is greater in developing nations and indigenous populations where the associated hearing loss contributes to poor education and employment outcomes. Current treatment and prevention is largely focused on vaccination and antibiotics. However, rates of OM, particularly in indigenous populations, remain high. With growing concerns regarding antibiotic resistance and antibiotic-associated complications, an alternative, more effective treatment is required. Administration of probiotics, both locally and systemically have been investigated for their ability to treat and prevent OM in children. This review explores the theoretical bases of probiotics, successful application of probiotics in medicine, and their use in the treatment and prevention of OM. We conclude that local administration of niche-specific probiotic bacteria that demonstrates the ability to inhibit the growth of otopathogens in vitro shows promise in the prevention and treatment of OM and warrants further investigation.

Educating and mobilizing youth to detect undiagnosed elevated blood pressure: searching for the silent killer.

BACKGROUND: Hispanic individuals with high blood pressure are less likely than other ethnic groups to be aware of their high blood pressure or to be on medication for the condition. We investigated the feasibility of using high school students in predominantly Hispanic neighborhoods to conduct a large-scale blood pressure screening and education outreach in their communities. METHODS: In 2005, 960 students from Los Angeles high schools in predominantly Hispanic communities were trained to conduct blood pressure screening and provide educational materials and referrals. A multivariable analysis using logistic regression was conducted to analyze the association between self-reported cardiovascular risk factors and elevated blood pressure. RESULTS: Students presented educational materials to 5395 persons and screened 5165 persons in their communities. Of 5395 individuals screened, 299 (6%) were found to have elevated blood pressure, of which only 77 (26%) were taking antihypertensive medication. Of those with elevated blood pressure on screening, 46% indicated they had a history of hypertension, and 3% of the entire screened community were identified as having elevated blood pressure for the first time. Older age, male sex, heavy alcohol consumption, and history of hypertension were all independently associated with elevated blood pressure. CONCLUSIONS: Training high school students to identify persons with elevated blood pressure is feasible and could reach large numbers of ethnic minorities unaware of their blood pressure status.

Sudden death in toddlers caused by influenza B infection: a report of two cases and a review of the literature.

Sudden fatal cases of influenza B infection in a 4-year-old girl and a 2-year-old boy are presented. Both children complained of abdominal pain without respiratory, neurologic or cardiac symptoms; additionally the girl had vomiting within 2 days of death. Autopsy revealed histological changes in the respiratory system consistent with a viral infection. Influenza B infection was identified by immunohistochemistry in the girl and real-time polymerase chain reaction in the boy. Additional testing including cultures, toxicology, and screening for metabolic disorders were negative. These cases illustrate the usefulness of viral testing, especially for influenza, in the medical legal autopsy of children even when the classic respiratory symptoms of flu are lacking.

The unsolved problem of otitis media in indigenous populations: a systematic review of upper respiratory and middle ear microbiology in indigenous children with otitis media.

BACKGROUND: Otitis media (OM) imposes a great burden of disease in indigenous populations around the world, despite a variety of treatment and prevention programs. Improved understanding of the pathogenesis of OM in indigenous populations is required to advance treatment and reduce prevalence. We conducted a systematic review of the literature exploring the upper airway and middle ear microbiota in relation to OM in indigenous children. METHODS: Papers targeting microbiota in relation to OM in children < 18 years indigenous to Australia, New Zealand, North America, and Greenland were sought. MEDLINE, CINAHL, EMBASE, Cochrane Library, and Informit databases were searched using key words. Two independent reviewers screened titles, abstracts, and then full-text papers against inclusion criteria according to PRISMA guidelines. RESULTS: Twenty-five papers considering indigenous Australian, Alaskan, and Greenlandic children were included. There were high rates of nasopharyngeal colonization with the three main otopathogens (Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis) in indigenous children with OM. Middle ear samples had lower rates of otopathogen detection, although detection rates increased when molecular methods were used. Pseudomonas aeruginosa and Staphylococcus aureus were commonly detected in middle ear discharge of children with chronic suppurative OM. There was a significant heterogeneity between studies, particularly in microbiological methods, which were largely limited to culture-based detection of the main otopathogens. CONCLUSIONS: There are high rates of otopathogen colonization in indigenous children with OM. Chronic suppurative OM appears to be associated with a different microbial profile. Beyond the main otopathogens, the data are limited. Further research is required to explore the entire upper respiratory tract/middle ear microbiota in relation to OM, with the inclusion of healthy indigenous peers as controls.

Does early communication mediate the relationship between motor ability and social function in children with cerebral palsy?

BACKGROUND: Children diagnosed with neurodevelopmental conditions such as cerebral palsy (CP) are at risk of experiencing restrictions in social activities negatively impacting their subsequent social functioning. Research has identified motor and communication ability as being unique determinants of social function capabilities in children with CP, to date, no research has investigated whether communication is a mediator of the relationship between motor ability and social functioning. AIMS: To investigate whether early communication ability at 24 months corrected age (ca.) mediates the relationship between early motor ability at 24 months ca. and later social development at 60 months ca. in a cohort of children diagnosed with cerebral palsy (CP). METHOD: A cohort of 71 children (43 male) diagnosed with CP (GMFCS I=24, 33.8%, II=9, 12.7%, III=12, 16.9%, IV=10, 14.1%, V=16, 22.5%) were assessed at 24 and 60 months ca. Assessments included the Gross Motor Function Measure (GMFM), the Communication and Symbolic Behaviour Scales-Developmental Profile (CSBS-DP) Infant-Toddler Checklist and the Paediatric Evaluation of Disability Inventory (PEDI). A mediation model was examined using bootstrapping. RESULTS: Early communication skills mediated the relationship between early motor abilities and later social functioning, b=0.24 (95% CI=0.08-0.43 and the mediation model was significant, F (2, 68)=32.77, p<0.001, R(2)=0.49. CONCLUSIONS AND IMPLICATION: Early communication ability partially mediates the relationship between early motor ability and later social function in children with CP. This demonstrates the important role of early communication in ongoing social development. Early identification of communication delay and enriched language exposure is crucial in this population.