RESUMO
BACKGROUND: New York State (NYS) utilizes a three-tiered cystic fibrosis newborn screening (CFNBS) algorithm that includes cystic fibrosis transmembrane conductance regulator (CFTR) gene sequencing. Infants with >1 CFTR variant of potential clinical relevance, including variants of uncertain significance or varying clinical consequence are referred for diagnostic evaluation at NYS cystic fibrosis (CF) Specialty Care Centers (SCCs). AIMS: As part of ongoing quality improvement efforts, demographic, screening, diagnostic, and clinical data were evaluated for 289 CFNBS-positive infants identified in NYS between December 2017 and November 2020 who did not meet diagnostic criteria for CF and were classified as either: CFTR-related metabolic syndrome/CF screen positive, inconclusive diagnosis (CRMS/CFSPID) or CF carriers. RESULTS: Overall, 194/289 (67.1%) had CFTR phasing to confirm whether the infant's CFTR variants were in cis or in trans. Eighteen complex alleles were identified in cis; known haplotypes (p.R117H+5T, p.F508del+p.L467F, and p.R74W+p.D1270N) were the most common identified. Thirty-two infants (16.5%) with all variants in cis were reclassified as CF carriers rather than CRMS/CFSPID. Among 263 infants evaluated at an NYS SCC, 70.3% were reported as having received genetic counseling about their results by any provider, with 96/263 (36.5%) counseled by a certified genetic counselor. CONCLUSION: Given the particularly complex genetic interpretation of results generated by CFNBS algorithms including sequencing analysis, additional efforts are needed to ensure families of infants with a positive CFNBS result have CFTR phasing when needed to distinguish carriers from infants with CRMS/CFSPID, and access to genetic counseling to address implications of CFNBS results.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Aconselhamento Genético , Genótipo , Triagem Neonatal , Pais , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , New York , Fibrose Cística/genética , Recém-Nascido , Masculino , Feminino , Triagem Neonatal/métodos , Fenótipo , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , LactenteAssuntos
Síndrome Congênita de Insuficiência da Medula Óssea , Humanos , Recém-Nascido , New York , Seguimentos , Feminino , MasculinoRESUMO
Elexacaftor, tezacaftor, ivacaftor (ETI) have been associated with marked clinical improvements in adults with CF, which appears to be associated with increased fertility. However, maternal and fetal effects of therapy continued during pregnancy are not well understood. We collected maternal blood, infant blood, cord blood, and breast milk from 3 mother-infant pairs from women who elected to remain on ETI therapy while pregnant. Our results demonstrated relatively high levels of ETI in cord blood, suggesting placental transfer of these compounds, as well as low levels of ETI in breast milk and infant blood, suggesting further transfer of these compounds to breast-fed infants in the postnatal period. These data underscore the need for larger studies on the effects of modulator surrounding reproduction.
Assuntos
Agonistas dos Canais de Cloreto , Fibrose Cística , Exposição Materna , Mães , Adulto , Aminofenóis , Benzodioxóis , Agonistas dos Canais de Cloreto/administração & dosagem , Fibrose Cística/tratamento farmacológico , Combinação de Medicamentos , Feminino , Humanos , Indóis , Lactente , Placenta , Gravidez , Pirazóis , Piridinas , Pirrolidinas , QuinolonasAssuntos
Aminofenóis/uso terapêutico , Benzodioxóis/uso terapêutico , Fibrose Cística/diagnóstico , Fibrose Cística/tratamento farmacológico , Indóis/uso terapêutico , Triagem Neonatal/métodos , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Quinolinas/uso terapêutico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Diagnóstico Diferencial , Combinação de Medicamentos , Feminino , Humanos , Recém-Nascido , GravidezRESUMO
Bacteria can survive antibiotic treatment both by acquiring antibiotic resistance genes and through mechanisms of tolerance that are based on phenotypic changes and the formation of metabolically inactive cells. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) that was isolated from a cystic fibrosis patient and had no increase in resistance but extremely high-level tolerance to ampicillin, vancomycin, and tetracycline. Specifically, the percentages of cells that survived 3.5-h antibiotic treatment (at 100 µg · ml-1) were 25.4% ± 4.3% and 51.9% ± 4.0% for ampicillin and tetracycline, respectively; vancomycin did not exhibit any significant killing. Consistent with the changes in antibiotic susceptibility, UM001B was found to have reduced penetration of ampicillin and vancomycin and accumulation of tetracycline compared to the reference strain ATCC 29212. Based on whole-genome sequencing, four amino acid substitutions were identified in one of the tetracycline efflux pump repressors (TetRs), compared to ATCC 29212. Results of molecular simulations and experimental assays revealed that these mutations could lead to higher levels of tetracycline efflux activity. Consistently, replicating these mutations in Escherichia coli MG1655 increased its tolerance to tetracycline. Overall, these findings provide new insights into the development of multidrug tolerance in E. faecalis, which can facilitate future studies to better control enterococcal infections.IMPORTANCEEnterococcus faecalis represents a major group of pathogens causing nosocomial infections that are resistant to multiple classes of antibiotics. An important challenge associated with E. faecalis infection is the emergence of multidrug-tolerant strains, which have normal MICs but do not respond to antibiotic treatment. Here, we report a strain of E. faecalis that was isolated from a cystic fibrosis patient and demonstrated high-level tolerance to ampicillin, vancomycin, and tetracycline. Whole-genome sequencing revealed critical substitutions in one of the tetracycline efflux pump repressors that are consistent with the increased tolerance of E. faecalis UM001B to tetracycline. These findings provide new information about bacterial antibiotic tolerance and may help develop more effective therapeutics.
Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Enterococcus faecalis/efeitos dos fármacos , Tetraciclina/farmacologia , Vancomicina/farmacologia , Enterococcus faecalis/genética , Testes de Sensibilidade MicrobianaRESUMO
Aberrant right subclavian artery with a left aortic arch is rare, but it is the most common congenital aortic arch anomaly. It can present as an incidental finding later in life or be symptomatic at a young age. Here, we describe a case of an aberrant right subclavian artery discovered in a 4 month old with respiratory distress and feeding difficulties. She underwent an extensive aerodigestive evaluation including bronchoscopy, both flexible and rigid, upper GI endoscopy, modified barium swallow with esophageal sweep, chest imaging, CT thorax and echocardiogram. The final decision per the management team was to observe the patient in order to allow more growth. She ultimately improved with age and remains asymptomatic.
RESUMO
DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.
Assuntos
Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Proteínas de Fluorescência Verde/metabolismo , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Ligação Competitiva , Cálcio/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Íleo/citologia , Íleo/efeitos dos fármacos , Íleo/enzimologia , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/citologia , Músculo Liso/enzimologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Fosforilação , Cultura Primária de Células , Inibidores de Proteínas Quinases/química , Proteômica , Pirazóis/química , Pirimidinonas/química , Coelhos , Proteínas Recombinantes de Fusão/genéticaRESUMO
Vascular injury and remodeling are common pathological sequelae of hypertension. Previous studies have suggested that the renin-angiotensin system acting through the type 1 angiotensin II (AT(1)) receptor promotes vascular pathology in hypertension. To study the role of AT(1) receptors in this process, we generated mice with cell-specific deletion of AT(1) receptors in vascular smooth muscle cells using Cre/Loxp technology. We crossed the SM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Agtr1a gene (Agtr1a (flox)), encoding the major murine AT(1) receptor isoform (AT(1A)). In SM22α-Cre(+)Agtr1a (flox/flox) (SMKO) mice, AT(1A) receptors were efficiently deleted from vascular smooth muscle cells in larger vessels but not from resistance vessels such as preglomerular arterioles. Thus, vasoconstrictor responses to angiotensin II were preserved in SMKO mice. To induce hypertensive vascular remodeling, mice were continuously infused with angiotensin II for 4 weeks. During infusion of angiotensin II, blood pressures increased significantly and to a similar extent in SMKO and control mice. In control mice, there was evidence of vascular oxidative stress indicated by enhanced nitrated tyrosine residues in segments of aorta; this was significantly attenuated in SMKO mice. Despite these differences in oxidative stress, the extent of aortic medial expansion induced by angiotensin II infusion was virtually identical in both groups. Thus, vascular AT(1A) receptors promote oxidative stress in the aortic wall but are not required for remodeling in angiotensin II-dependent hypertension.
Assuntos
Aorta/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Análise de Variância , Angiotensina II/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Hipertensão/induzido quimicamente , Hipertensão/patologia , Hipertensão/fisiopatologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasoconstritores/farmacologiaRESUMO
Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30-40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.
Assuntos
Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Músculo Estriado/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfoproteínas/metabolismo , Adulto , Animais , Western Blotting , Núcleo Celular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Mutantes , Microscopia Confocal , Proteínas Musculares/genética , Quinase de Cadeia Leve de Miosina/genética , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas/genética , Fosforilação , Gravidez , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Útero/metabolismoRESUMO
In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1(-/-) mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to alpha-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1(-/-) mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1(+/+) mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas Musculares/biossíntese , Proteínas Musculares/fisiologia , Músculo Liso Vascular/metabolismo , Fosfoproteínas/genética , Animais , Feminino , Deleção de Genes , Humanos , Camundongos , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Miosinas/metabolismo , Fenótipo , Fosfoproteínas/fisiologia , Fosforilação , Condicionamento Físico AnimalRESUMO
Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.