43 research outputs found
Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis
Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (Δ) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Îζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class.Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria.Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial Δ, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from Δ to ζ and that ζ, Δ, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed
Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study
Summary
Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally.
Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies
have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of
the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income
countries globally, and identified factors associated with mortality.
Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to
hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis,
exomphalos, anorectal malformation, and Hirschsprungâs disease. Recruitment was of consecutive patients for a
minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical
status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary
intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause,
in-hospital mortality for all conditions combined and each condition individually, stratified by country income status.
We did a complete case analysis.
Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital
diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal
malformation, and 517 with Hirschsprungâs disease) from 264 hospitals (89 in high-income countries, 166 in middleincome
countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male.
Median gestational age at birth was 38 weeks (IQR 36â39) and median bodyweight at presentation was 2·8 kg (2·3â3·3).
Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income
countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups).
Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome
countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries;
pâ€0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients
combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88â4·11],
p<0·0001; middle-income vs high-income countries, 2·11 [1·59â2·79], p<0·0001), sepsis at presentation (1·20
[1·04â1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention
(ASA 4â5 vs ASA 1â2, 1·82 [1·40â2·35], p<0·0001; ASA 3 vs ASA 1â2, 1·58, [1·30â1·92], p<0·0001]), surgical safety
checklist not used (1·39 [1·02â1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed
(ventilation 1·96, [1·41â2·71], p=0·0001; parenteral nutrition 1·35, [1·05â1·74], p=0·018). Administration of
parenteral nutrition (0·61, [0·47â0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65
[0·50â0·86], p=0·0024) or percutaneous central line (0·69 [0·48â1·00], p=0·049) were associated with lower mortality.
Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome,
middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will
be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger
than 5 years by 2030
Immunolocalization of the Na\u3csup\u3e+\u3c/sup\u3eâK\u3csup\u3e+\u3c/sup\u3eâ2Cl\u3csup\u3e-\u3c/sup\u3e Cotransporter in Peripheral Nervous Tissue of Vertebrates
Efflux of Clâ through GABAA-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Clâ inside sensory neurons occurs through an Na+âK+â2Clâ cotransport system that generates and maintains the electrochemical gradient for this outward Clâ current. We studied the immunolocalization of the Na+âK+â2Clâ cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at âŒ160 kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to âŒ135 kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwan cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and ÎČ-mercaptoethanol also uncovered intense cotransporter immunoreactivity in SchmidtâLanterman incisures at the light microscopic level. The localization of the Na+âK+â2Clâcotransporter protein is consistent with its function as a Clâ-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K+ uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons
Immunolocalization of the Na\u3csup\u3e+\u3c/sup\u3eâK\u3csup\u3e+\u3c/sup\u3eâ2Cl\u3csup\u3e-\u3c/sup\u3e Cotransporter in Peripheral Nervous Tissue of Vertebrates
Efflux of Clâ through GABAA-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Clâ inside sensory neurons occurs through an Na+âK+â2Clâ cotransport system that generates and maintains the electrochemical gradient for this outward Clâ current. We studied the immunolocalization of the Na+âK+â2Clâ cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at âŒ160 kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to âŒ135 kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwan cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and ÎČ-mercaptoethanol also uncovered intense cotransporter immunoreactivity in SchmidtâLanterman incisures at the light microscopic level. The localization of the Na+âK+â2Clâcotransporter protein is consistent with its function as a Clâ-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K+ uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons
Immunolocalization of the Na\u3csup\u3e+\u3c/sup\u3eâK\u3csup\u3e+\u3c/sup\u3eâ2Cl\u3csup\u3e-\u3c/sup\u3e Cotransporter in Peripheral Nervous Tissue of Vertebrates
Efflux of Clâ through GABAA-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Clâ inside sensory neurons occurs through an Na+âK+â2Clâ cotransport system that generates and maintains the electrochemical gradient for this outward Clâ current. We studied the immunolocalization of the Na+âK+â2Clâ cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at âŒ160 kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to âŒ135 kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwan cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and ÎČ-mercaptoethanol also uncovered intense cotransporter immunoreactivity in SchmidtâLanterman incisures at the light microscopic level. The localization of the Na+âK+â2Clâcotransporter protein is consistent with its function as a Clâ-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K+ uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons
Enzymatically cross-linked arabinoxylan microspheres as oral insulin delivery system
Arabinoxylans (AX) microspheres with different insulin/AX mass ratio were prepared by formation of phenoxy radical issued from the ferulic acid by enzymatic oxidation (entrapped in situ of insulin). Phenolic acid content and FT-IR spectrum of unloaded and insulin-loaded AX microspheres revealed that the phenoxy radical issued from the ferulic acid by enzymatic oxidation did not interact covalently with insulin. The microspheres showed a spherical shape, smooth surface and an average diameter of particles of 320âŻÎŒm. In vitro control release found that AX microspheres minimized the insulin loss in the upper GI tract, retaining high percentage (~75%) of insulin in its matrix. The stability of the secondary structure of insulin was studied by dichroism circular (CD). The CD spectra of insulin released from AX microspheres did not change according to the insulin/AX mass ratio of the microsphere. Significant hypoglycemic effects with improved insulin-relative bioavailability tested on an in vivo murine model revealed the efficacy of these enzymatically cross-linked arabinoxylans microspheres as a new oral insulin carrier
Paclitaxel and cisplatin versus paclitaxel and cisplatin plus all-trans retinoic acid for the prevention of chemotherapy-induced neuropathy: A randomized phase II trial
The Absence of Heat Shock Protein HSP101 Affects the Proteome of Mature and Germinating Maize Embryos
Maize heat shock protein HSP101 accumulates during embryo
maturation
and desiccation and persists at high levels during the first 24 h
following kernel imbibition in the absence of heat stress. This protein
has a known function in disaggregation of high molecular weight complexes
and has been proposed to be a translational regulator of specific
mRNAs. Here, a global proteomic approach was used to identify changes
in the maize proteome due to the absence of HSP101 in embryos from
mature-dry or 24 h-imbibed kernels. A total of 26 protein spots from
the mature dry embryo exhibited statistically significant expression
changes in the L10 inbred <i>hsp101</i> mutant (<i>hsp101-m5::Mu1</i>/<i>hsp101-m5::Mu1</i>) line as
compared to the corresponding wild type (<i>Hsp101</i>/<i>Hsp101</i>). Additional six spots reproducibly showed qualitative
changes between the mutant and wild-type mature and germinating embryos.
Several chaperones, translation-related proteins, actin, and enzymes
participating in cytokinin metabolism were identified in these spots
by tandem mass-spectrometry (MS). The proteomic changes partially
explain the altered root growth and architecture observed in young <i>hsp101</i> mutant seedlings. In addition, specific protein de
novo synthesis was altered in the 24 h-imbibed mutant embryos indicating
that maize HSP101 functions as both chaperone and translational regulator
during germination. Supporting this, HSP101 was found as part of Cap-binding
and translation initiation complexes during early kernel imbibition.
Overall, these findings expose the relevance of maize HSP101 for protein
synthesis and balance mechanisms during germination
The Absence of Heat Shock Protein HSP101 Affects the Proteome of Mature and Germinating Maize Embryos
Maize heat shock protein HSP101 accumulates during embryo
maturation
and desiccation and persists at high levels during the first 24 h
following kernel imbibition in the absence of heat stress. This protein
has a known function in disaggregation of high molecular weight complexes
and has been proposed to be a translational regulator of specific
mRNAs. Here, a global proteomic approach was used to identify changes
in the maize proteome due to the absence of HSP101 in embryos from
mature-dry or 24 h-imbibed kernels. A total of 26 protein spots from
the mature dry embryo exhibited statistically significant expression
changes in the L10 inbred <i>hsp101</i> mutant (<i>hsp101-m5::Mu1</i>/<i>hsp101-m5::Mu1</i>) line as
compared to the corresponding wild type (<i>Hsp101</i>/<i>Hsp101</i>). Additional six spots reproducibly showed qualitative
changes between the mutant and wild-type mature and germinating embryos.
Several chaperones, translation-related proteins, actin, and enzymes
participating in cytokinin metabolism were identified in these spots
by tandem mass-spectrometry (MS). The proteomic changes partially
explain the altered root growth and architecture observed in young <i>hsp101</i> mutant seedlings. In addition, specific protein de
novo synthesis was altered in the 24 h-imbibed mutant embryos indicating
that maize HSP101 functions as both chaperone and translational regulator
during germination. Supporting this, HSP101 was found as part of Cap-binding
and translation initiation complexes during early kernel imbibition.
Overall, these findings expose the relevance of maize HSP101 for protein
synthesis and balance mechanisms during germination