Excessive gas exchange impairment during exercise in a subject with a history of bronchopulmonary dysplasia and high altitude pulmonary edema

A 27-year-old male subject (V(O2 max)), 92% predicted) with a history of bronchopulmonary dysplasia (BPD) and a clinically documented case of high altitude pulmonary edema (HAPE) was examined at rest and during exercise. Pulmonary function testing revealed a normal forced vital capacity (FVC, 98.1% predicted) and diffusion capacity for carbon monoxide (D(L(CO)), 91.2% predicted), but significant airway obstruction at rest [forced expiratory volume in 1 sec (FEV(1)), 66.5% predicted; forced expiratory flow at 50% of vital capacity (FEF(50)), 34.3% predicted; and FEV(1) /FVC 56.5%] that was not reversible with an inhaled bronchodilator. Gas exchange worsened from rest to exercise, with the alveolar to arterial P(O2) difference (AaD(O2)) increasing from 0 at rest to 41 mmHg at maximal normoxic exercise (VO(2) = 41.4 mL/kg/min) and from 11 to 31 mmHg at maximal hypoxic exercise (VO(2) = 21.9 mL/kg/min). Arterial P(O2) decreased to 67.8 and 29.9 mmHg at maximal normoxic and hypoxic exercise, respectively. These data indicate that our subject with a history of BPD is prone to a greater degree of exercise-induced arterial hypoxemia for a given VO(2) and F(I(O2)) than healthy age-matched controls, which may increase the subject's susceptibility to high altitude illness

A rotary mechanism for allostery in bacterial hybrid malic enzymes

This project was funded by BBSRC studentship 1500753 to C.J.H. and a BBSRC David Phillips fellowship to P.J.M. (BB/S010122/1).Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.Publisher PDFPeer reviewe

Activity of Bdellovibrio Hit Locus Proteins, Bd0108 and Bd0109, Links Type IVa Pilus Extrusion/Retraction Status to Prey-Independent Growth Signalling

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to preyindependent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation background

AltitudeOmics: exercise-induced supraspinal fatigue is attenuated in healthy humans after acclimatization to high altitude

Aims: We asked whether acclimatisation to chronic hypoxia (CH) attenuates the level of supraspinal fatigue that is observed after locomotor exercise in acute hypoxia (AH). Methods: Seven recreationally-active participants performed identical bouts of constant-load cycling (131±39W, 10.1±1.4min) on three occasions: 1) in normoxia (N, PIO2, 147.1mmHg); 2) in AH (FIO2, 0.105; PIO2, 73.8mmHg); 3) after 14 days in CH (5,260m; PIO2, 75.7mmHg). Throughout trials, prefrontal-cortex tissue oxygenation and middle cerebral artery blood velocity (MCAV) were assessed using near-infrared-spectroscopy and transcranial Doppler sonography. Pre- and post-exercise twitch responses to femoral nerve stimulation and transcranial magnetic stimulation were obtained to assess neuromuscular and corticospinal function. Results: In AH, prefrontal oxygenation declined at rest (Δ7±5%) and end-exercise (Δ26±13) (P<0.01); the degree of deoxygenation in AH was greater than N and CH (P<0.05). The cerebral O2 delivery index (MCAv×CaO2) was 19±14% lower during the final minute of exercise in AH compared to N (P=0.013) and 20±12% lower compared to CH (P=0.040). Maximum voluntary and potentiated twitch force were decreased below baseline after exercise in AH and CH, but not N. Cortical voluntary activation decreased below baseline after exercise in AH (11%, P=0.014), but not CH (6%, P=0.174) or N (4%, P=0.298). A twofold greater increase in motor evoked potential amplitude was evident after exercise in CH compared to AH and N. Conclusion: These data indicate that exacerbated supraspinal fatigue after exercise in AH is attenuated after 14 days of acclimatisation to altitude. The reduced development of supraspinal fatigue in CH may have been attributable to increased corticospinal excitability, consequent to an increased cerebral O2 delivery

Predation of HI strains compared to predatory HD100.

<div><p>Time-lapse microscopy of individual wild-type HD100 cells (<b>A</b>(<b>a</b>)) and cells of the markerless deletion mutant of <i>bd0108</i> (A(b)) preying upon the <i>E. coli</i> strain pMAL-p2_mCherry with a fluorescent periplasm. At T=0 there is attachment of free swimming <i>Bdellovibrio</i> in both strains, with invasion occurring within 2 frames (5 minutes) for most cells observed. Arrows indicate both free swimming and attached <i>Bdellovibrio</i> cells. By 1 hour the <i>Bdellovibrio</i> is established in the periplasm and begun to grow as a filament. By 8 hours the growing filaments of both the wild-type and deletion strains have septated to form single progeny and the prey bursts releasing them. Predation was carried out on a 1% Agarose pad with images acquired every 2.5 minutes.</p> <p><b>B</b>. Reduction of <i>E. coli</i> numbers in a predatory lysate comparing wild-type HD100 or spontaneously generated HIs with the markerless deletion of <i>bd0108</i> strains. The spontaneously generated HI strains included some with a variety of point mutations or the common 42bp deletion in <i>bd0108</i> and some with a wild-type copy of <i>bd0108</i>. HI cultures readily prey upon <i>E. coli</i> in liquid cultures, reducing prey numbers by several logs in 2 days with predation by the population as a whole appearing to be slightly slower than wild-type HD100. All HI strains were grown axenically, independently of prey cells before the experiment. </p></div

RT-PCR expression of the sRNA downstream of <i>bd0108</i> and expression of a control gene <i>dnaK</i>.

<p>Expression of a small, non-coding RNA downstream of <i>bd0108</i> was shown to be different in HI strains carrying different mutations in <i>bd0108</i> and attack phase HD100. Expression was highest in HID13 (ATA->ATG start codon mutation) and the strains with the markerless deletion of <i>bd0108</i>. Strain HID22 (<i>bd0108∆</i>42bp), had slightly lower expression, while those strains with a wild-type <i>bd0108</i>; HD100 and HID2, show much lower expression of the sRNA. Expression of <i>dnaK</i> was uniform across the samples indicating a matched amount of total RNA was used for the experiment.</p

PFU to CFU ratio for pooled spontaneous HI strains and the markerless <i>bd0108</i> deletion HI mutants of <i>Bdellovibrio</i>.

<p>In the spontaneous generated HI strains; HID2 and HID26 (wild-type for <i>bd0108</i>) HID6, HID13, HID18, (point mutations in <i>bd0108</i>) and HID22 (<i>bd0108∆</i>42bp), and the markerless deletion mutants of <i>bd0108</i>, complementation tests shows that these strains carrying the pSUP404.2 plasmid encoding the wild-type <i>bd0108</i> is significantly deficient in the capacity to form HI colonies on PY agar plates compared to the capacity to form plaques on double layer overlay plates. This was significant compared to cells containing the pSUP404.2 plasmid alone, or the pSUP404.2-Δ42 with the 42 bp deletion variant of <i>bd0108</i>. The ratio of CFU:PFU was reduced from 2.16 x 10^-1 in strains containing the pSUP404.2 alone to 3.21 x 10^-2 in strains containing pSUP404.2-108 (p = 0.026) but were not significantly reduced relative to strains containing pSUP404.2-Δ42, 2.56 x 10^-1 (p = 0.31). The difference between strains carrying the pSUP404.2-108 and the pSUP404.2-Δ42 was also significant (P<<0.01).</p

Model for possible interactions of Bd0108/Bd0109 controlling the extrusion and retraction of pili.

<div><p>A. Operonal structure of the <i>bd0108 </i><i>hit</i> locus and surrounding genes, predicted to have a role in the formation of a Type IVb pilus. Genes are colour coded to correspond to their predicted function in the pilus diagrams underneath.</p> <p><b>B</b>. In wild-type cells <i>bd0108</i> and <i>bd0109</i> are co-expressed, the mRNA is then translated into proteins containing a signal sequence recognised by the Sec system, the signal is cleaved, and the proteins are transported into the periplasm where the mature Bd0108 protein transiently interacts with Bd0109 to sequester it. When Bd0109 is unbound, it could then anchor at either the cell wall, or with the mature pilus fibre. Both scenarios are possible due to Bd0109’s structural cleft binding carbohydrate that is present in both cell wall and the mature and glycosylated pili. Bd0109 mediates successful pilus extrusion/retraction and signal back into the cytoplasm. In wild-type pilus formation Bd1290 pre-pilins are held in the inner membrane and are assembled into the pilus fibre possibly by the flp pilus ATPases Bd0110 and Bd0111. The balance of sequestering and release of Bd0109 by Bd0108 in the periplasm permits to successful extrusion and retraction of the pilus fibre upon environmental cues.</p> <p><b>C</b>. In the absence of Bd0108 protein, Bd0109 is not sequestered and is free to mediate more frequently with pilus extrusion and retraction, resulting in very few pili extruded beyond the cell surface and cues for HI growth signalled to the cell.</p> <p><b>D</b>. In HI strains containing the 42 bp deletion variant of <i>bd0108</i>, the gene is still expressed. The truncated form of Bd0108 alters the dynamics of the Bd0109 functionalisation are altered (possibly by over-sequestration of Bd0109) and hyper-extruded pili are seen on the surface more frequently. Hyper-extruded pili or no pili send similar internal signals to regulate prey independent growth.</p></div