Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.     

Novel Arenavirus Sequences in Hylomyscus sp. and Mus (Nannomys) setulosus from C?te d'Ivoire: Implications for Evolution of Arenaviruses in Africa

This study aimed to identify new arenaviruses and gather insights in the evolution of arenaviruses in Africa. During 2003 through 2005, 1,228 small mammals representing 14 different genera were trapped in 9 villages in south, east, and middle west of Côte d''Ivoire. Specimens were screened by pan-Old World arenavirus RT-PCRs targeting S and L RNA segments as well as immunofluorescence assay. Sequences of two novel tentative species of the family Arenaviridae, Menekre and Gbagroube virus, were detected in Hylomyscus sp. and Mus (Nannomys) setulosus, respectively. Arenavirus infection of Mus (Nannomys) setulosus was also demonstrated by serological testing. Lassa virus was not found, although 60% of the captured animals were Mastomys natalensis. Complete S RNA and partial L RNA sequences of the novel viruses were recovered from the rodent specimens and subjected to phylogenetic analysis. Gbagroube virus is a closely related sister taxon of Lassa virus, while Menekre virus clusters with the Ippy/Mobala/Mopeia virus complex. Reconstruction of possible virus–host co-phylogeny scenarios suggests that, within the African continent, signatures of co-evolution might have been obliterated by multiple host-switching events.     

L’Inhibine B sérique, marqueur sensible de la production spermatique chez l’homme

Several recent papers indicate that the blood concentration of inhibin B measured by the dimeric assay is a marker of human spermatogenesis. The aim of the present study was to validate the dimeric assay in our hospital. For this purpose, we studied a population of 106 patients attending our infertility clinic. We found that serum inhibin B levels were positively correlated with the level of sperm production as reflected by the sperm concentration and negatively correlated with serum FSH levels. Serum inhibin B concentrations were found to be superior to FSH levels for discriminating between subgroups of patients with different levels of sperm production. For example, the differences in the inhibin B levels in severe oligozoospermia (< 5×106 sperm/ml) compared to non-obstructive azoospermia were more pronounced than the differences in FSH (p=0.0002 vs p=0.007, respectively). Overall, the results obtained in the present study were similar to those reported in other studies based on the same assay. Serum inhibin B levels in patients with non obstructive azoospermia were ten times lower than in patients with normal sperm concentrations. From a practical point of view, the measurement of serum inhibin B opens up new possibilities for the diagnosis and prognosis of many testicular disorders. Firstly, serum inhibin B determination should be performed whenever reproductive hormones are prescribed. In the particular case of azoospermia, serum inhibin B concentrations appear to be particularly useful to distinguish between obstructive and non-obstructive situations. Finally, according to a recent publication, a serum inhibin B cut-off value (> 40 pg/mL) could predict the success of testicular sperm extraction in patients with non-obstructive azoospermia. Serum inhibin B could therefore represent a unique non-invasive marker of focal hypospermatogenesis in men with non-obstructive azoospermia, who are candidates for intracytoplasmic sperm injection (ICSI) and a useful early marker to monitor possible recovery of spermatogenesis after chemotherapy or radiotherapy.     

Mutation analysis of NR5A1 encoding steroidogenic factor 1 in 77 patients with 46, XY disorders of sex development (DSD) including hypospadias

Background

Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias.

Methodology/Principal Findings

We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein.

Conclusions/Significance

Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations.     

Severe Nocturnal and Postexercise Hypoxia in Children and Adolescents with Sickle Cell Disease

Hypoxia is a common feature in children with sickle cell disease (SCD) that is inconsistently associated with painful crises and acute chest syndrome. To assess the prevalence and risk factors of hypoxia, we recorded daytime, nocturnal, and postexercise pulse oximetry (SpO₂) values in 39 SCD patients with a median age of 10.8 years. Median daytime SpO₂ was 97% (range, 89%–100%), and 36% of patients had daytime hypoxia defined as SpO₂<96%. Median nocturnal SpO₂ was 94.7% (range, 87.7%–99.5%), 50% of patients had nocturnal hypoxia defined as SpO₂≤93%, and 11(37%) patients spent more than 10% of their total sleep time with SpO₂<90%. Median postexercise SpO₂ was 94% (range, 72%–100%) and 44.7% of patients had postexercise hypoxia defined as an SpO₂ decrease ≥3% after a 6-minute walk test. Among patients with normal daytime SpO₂, 35% had nocturnal and 42% postexercise hypoxia. Compared to 9 patients without daytime, nocturnal, or postexercise hypoxia, 25 patients with hypoxia under at least one of these three conditions had greater anemia severity (P = 0.01), lower HbF levels (P = 0.04), and higher aspartate aminotransferase levels (P = 0.03). Males predominated among patients with postexercise hypoxia (P = 0.004). Hypoxia correlated neither with painful crises nor with acute chest syndrome. Of 32 evaluable patients, 6 (18.8%) had a tricuspid regurgitation velocity ≥2.6 m/s, and this feature was associated with anemia (P = 0.044). Median percentage of the predicted distance covered during a 6-minute walk test was 86% [46–120]; the distance was negatively associated with LDH (P = 0.044) and with a past history of acute chest syndrome (P = 0.009). In conclusion, severe episodes of nocturnal and postexercise hypoxia are common in children with SCD, even those with normal daytime SpO₂.     

Smartphone and a freely available application as a new tool to record locomotor activity rhythm in large mammals and humans

ABSTRACT

Daily pattern of locomotor activity (LA), one of the most studied rhythms in humans and rodents, has not been widely investigated in large mammals. This is partly due to the high cost and breakability of used automatic devices. Since last decade, smartphones are becoming ubiquitous. Meanwhile, several applications detecting activity by using internal sensors were made available. In this study, we assumed that this device could be a cheaper and easier way to measure the LA rhythm in humans and large mammals, like camel and goat. A smartphone application (Nokia Mate Health), normally used to quantify physical activities in humans, was chosen for the study. To validate the rhythm data obtained from the smartphone, LA rhythm was simultaneously recorded using an automatic device, the Actiwatch-Mini®. Results showed that the smartphone provided a clear and significant daily rhythm of LA. The visual assessment of the superimposed LA rhythm’s curves in all three species showed that the smartphone application displayed similar rhythms as those recorded by the Actiwatch-Mini. Highly significant positive correlation (p≤ 0.0001) exists between the two recording rhythms. The daily periods were both the same at 24.0 h. Acrophases were also significantly similar and occurring around mid-day: 11:40 ± 0.35 h vs 11:41 ± 0.35 h for the camel, 11:25 ± 0.19 h vs 11:37 ± 0.25 h for the goat and 13:04 ± 0.11 h vs 13:51 ± 0.28 h for humans using smartphone and Actiwatch, respectively. The related mesor and amplitude were also close between the two recording devices. Results indicate clearly that using smartphones constitutes a reliable cheap tool to study LA rhythm for chronobiology studies, especially in laboratories facing lack of funding.     

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.     

Novel arenavirus sequences in Hylomyscus sp. and Mus (Nannomys) setulosus from Côte d'Ivoire: implications for evolution of arenaviruses in Africa

This study aimed to identify new arenaviruses and gather insights in the evolution of arenaviruses in Africa. During 2003 through 2005, 1,228 small mammals representing 14 different genera were trapped in 9 villages in south, east, and middle west of C?te d'Ivoire. Specimens were screened by pan-Old World arenavirus RT-PCRs targeting S and L RNA segments as well as immunofluorescence assay. Sequences of two novel tentative species of the family Arenaviridae, Menekre and Gbagroube virus, were detected in Hylomyscus sp. and Mus (Nannomys) setulosus, respectively. Arenavirus infection of Mus (Nannomys) setulosus was also demonstrated by serological testing. Lassa virus was not found, although 60% of the captured animals were Mastomys natalensis. Complete S RNA and partial L RNA sequences of the novel viruses were recovered from the rodent specimens and subjected to phylogenetic analysis. Gbagroube virus is a closely related sister taxon of Lassa virus, while Menekre virus clusters with the Ippy/Mobala/Mopeia virus complex. Reconstruction of possible virus-host co-phylogeny scenarios suggests that, within the African continent, signatures of co-evolution might have been obliterated by multiple host-switching events.     

When Breathing Interferes with Cognition: Experimental Inspiratory Loading Alters Timed Up-and-Go Test in Normal Humans

Human breathing stems from automatic brainstem neural processes. It can also be operated by cortico-subcortical networks, especially when breathing becomes uncomfortable because of external or internal inspiratory loads. How the “irruption of breathing into consciousness” interacts with cognition remains unclear, but a case report in a patient with defective automatic breathing (Ondine''s curse syndrome) has shown that there was a cognitive cost of breathing when the respiratory cortical networks were engaged. In a pilot study of putative breathing-cognition interactions, the present study relied on a randomized design to test the hypothesis that experimentally loaded breathing in 28 young healthy subjects would have a negative impact on cognition as tested by “timed up-and-go” test (TUG) and its imagery version (iTUG). Progressive inspiratory threshold loading resulted in slower TUG and iTUG performance. Participants consistently imagined themselves faster than they actually were. However, progressive inspiratory loading slowed iTUG more than TUG, a finding that is unexpected with regard to the known effects of dual tasking on TUG and iTUG (slower TUG but stable iTUG). Insofar as the cortical networks engaged in response to inspiratory loading are also activated during complex locomotor tasks requiring cognitive inputs, we infer that competition for cortical resources may account for the breathing-cognition interference that is evidenced here.     

Proposal for the simultaneous measurement of light absorption and backscattering by aquatic particulates

The paper presents a new experimental method that measures lightabsorption and backscattering by aquatic particles, combiningthe innovative features of the ‘modified filter–transfer–freeze’(FTF) and ‘transmittance–reflectance’ (T-R)techniques, namely no path-length amplification and accuratecorrectionfor scattering loss, respectively. This method (in short: theFTF/T-R method) measures both the transmittance and the reflectanceof particles deposited on a glass slide, using a double-beamspectrophotometer equipped with an integrating-sphere attachment.The data are interpreted by an algorithm that yields both theabsorption and the backscattering coefficients of the particlesample. The paper includes a summary of results of validationtests, that have been performed using samples ranging from purephytoplankton (low light scattering) to pure detrital particles(high light scattering), as well as a detailed analysis of theexperimental error. The FTF/T-R measurement is somewhat morelaborious than the standard transmittance measurement of particlesretained on glass fibre filters. However, it has the importantasset of permitting the simultaneous determination of lightabsorption and backscattering and it yields more accurate absorptiondata in situations where the magnitude of path amplificationby glass fibre filters is uncertain and light scattering bythe particles is high.