Imaging of Bubonic Plague Dynamics by In Vivo Tracking of Bioluminescent Yersinia pestis

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Suivi in vivo et en temps réel du processus infectieux induit par Yersinia pestis

NCThe enterobacteria Yersinia pestis causes plague, a deadly infectious disease infamously known for millions of death caused during three major pandemics. The bacterium possesses virulence mechanisms that have not been completely elucidated. Mouse model has long been used for pathology studies, yet usual experimentation is time-consuming and laborious and requires large groups of animals. Bioluminescence imaging provides an efficient improvement in infection monitoring by providing non-invasive, real-time in vivo detection of living bacteria. We applied this technique to Y. pestis virulent strain CO92 and proved that this bacterium could emit detectable bioluminescence signals both in vitro and in vivo. Light intensity was correlated to cfu charges in spleen, liver and inguinal lymph node. By monitoring bubonic plague, we observed the sequence of bacterial colonization steps, from the injection site to the inguinal draining lymph node. The next target was the ipsilateral axillary lymph node: we proved that bacteria followed a direct path from the inguinal lymph node to the axillary lymph node. The bacteria then reached and colonized blood-filtering organs such as the spleen and the liver. In the latest times of the disease, septicemia was observed with a typical whole-body bioluminescence. Our results showed that the high variability in the kinetics of bubonic plague was attributable to the length of time during which Y. pestis remained confined in the injection site. Once the bacteria had reached the draining lymph nodes, the disease evolved in a very fast and regular way to septicemia within two days.Après trois pandémies majeures responsables de millions de morts, la peste n'a pas encore disparu. Cette maladie est causée par la bactérie Yersinia pestis, dont les mécanismes de virulence sont encore mal compris. Le suivi d'infection de la peste bubonique chez la souris, méthode classique pour étudier le processus infectieux, requiert beaucoup d'animaux et de temps pour obtenir des résultats significatifs. L'imagerie in vivo et en temps réel par bioluminescence permet de suivre la progression du pathogène au cours du processus infectieux en observant les animaux de façon non invasive. Nous avons transformé la souche virulente CO92 avec le plasmide pEm7-luxCDABE et confirmé la production de bioluminescence in vitro et in vivo. Nous avons pu quantifier la charge bactérienne dans plusieurs organes colonisés sans sacrifier l'animal et établir le schéma de progression de la bactérie au cours de la maladie. Après formation d'un foyer infectieux au site d'injection, la colonisation du ganglion lymphatique inguinal drainant ce site a été observée. Nous avons démontré que la bactérie suit un trajet direct du ganglion lymphatique inguinal au ganglion lymphatique axillaire. L'étape suivante est la colonisation des organes filtrant le sang, puis survient la septicémie dans les phases terminales de la mort. Nous avons établi que la forte variabilité dans le processus infectieux était due au temps pendant lequel la bactérie était contenue au site d'injection. À partir du moment où les ganglions lymphatiques sont colonisés, la cinétique de progression est à la fois régulière et rapide ; la septicémie survient dans les deux jours, suivie de près par la mort

Linking a bioluminescence signal to the colonization of a specific organ.

<p>(A) Example of the light emitted by a live animal. (B) Light emitted by the organs of the same animal. Arrows point at: (1) axillary lymph nodes, (2) liver (one lobe still in place and the remaining of the organ removed from its anatomical site), (3) injection site (split in two parts during dissection), (4) inguinal lymph nodes, and (5) spleen (removed from its anatomical site and visualized in live animals mostly in ventral position). On the dissection picture, the gut has been removed and placed next to the right hind leg, while the peritoneum including a portion of the infection site is located next to the left hind leg. Color bars on the right of each picture show the intensity of bioluminescence light coded in the picture from indigo to red.</p

Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery ▿

Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria

Bioluminescence imaging of <i>Y. pestis</i>.

<p><i>Y. pestis</i> CO92 or CO92(pLux) were grown for 48 h at 28°C on LB-Carb plates. The plates were placed in an IVIS 100 device dark chamber, photographed under normal light and then bioluminescence emission was detected using a 7 s exposure time and a picture binning of 4. Both pictures were superimposed by the Living image software. Colored dots represent bioluminescence emitting bacteria. The color bar on the right shows the intensity of bioluminescence light coded in the picture from indigo (4.1×10⁷ photon/s.cm².steradian) to red (6.9×10⁸ photon/s.cm².steradian).</p

Time of appearance of a bioluminescent signal in the various organs of infected mice.

<p>Each circle corresponds to an individual animal. Horizontal bars represent the mean and vertical bars the standard error of the mean of the data collected from 24 mice. LN: lymph node.</p

Different steps of bacterial spread during bubonic plague.

<p>The bioluminescent signal was visible first at the injection site (A). It then reached the inguinal lymph node (B), the axillary lymph node (C), the liver (D, dorsal position) and the spleen (D, ventral position), and finally the entire body (E). Each picture is an example of an animal displaying a signal characteristic of each step. The color scale on the right represents the settings used to monitor light emission in all mice throughout the observation period.</p

Lymphatic connection between the inguinal and axillary lymph nodes.

<p>(A) Observation of a bioluminescent signal connecting the two lymph nodes on a live and on a necropsied mouse. (B) Staining of the lymphatic system after injection in the <i>linea alba</i> of the Evans blue dye. The axillary and inguinal lymph nodes are stained in blue and the lymphatic vessel draining the two nodes is seen as a thin pale blue line adjacent to the blood vessels.</p

Establishment of cancer cell line originating from a patient with high-grade serous ovarian carcinoma

Aim: Ovarian cancer is a serious malignancy with high prevalence and mortality. Methods: We isolated and characterized an ovarian high-grade serous cancer cell line (M4) from a tumor of a Vietnamese patient with ovarian carcinoma. Results: The M4 cancer cell line showed good proliferation and stability in culture. Morphologically, the M4 cells showed similar characteristics to tumor cells such as a polyhedral shape, large irregular nuclei, high nuclear/cytoplasmic ratio, high nuclear density and expressing cancer markers like CA125, p53 and Ki67 markers. Conclusion: We have successfully isolated and characterized the M4 cell line from a Vietnamese patient with ovarian carcinoma