Computational hyperspectral light-sheet microscopy.

We describe a computational light-sheet microscope designed for hyperspectral acquisition at high spectral resolution. The fluorescence light emitted from the full field-of-view is focused along the entrance slit of an imaging spectrometer using a cylindrical lens. To acquire the spatial dimension orthogonal to the slit of the spectrometer, we propose to illuminate the specimen with a sequence of structured light patterns and to solve the image reconstruction problem. Beam shaping is obtained simply using a digital micromirror device in conjunction with a traditional selective plane illumination microscopy setup. We demonstrate the feasibility of this method and report the first results in vivo in hydra specimens labeled using two fluorophores.

Six-week antibiotic therapy after one-stage replacement arthroplasty for hip and knee periprosthetic joint infection.

OBJECTIVES: One-stage replacement arthroplasty for treatment of periprosthetic joint infection (PJI) results in similar cure rate than two-stage (around 85-92%), but antibiotic therapy duration is not well established. The aim of this study was to evaluate the efficacy of a short six-week antibiotic course in periprosthetic joint infections after onstage exchange. PATIENTS AND METHODS: Retrospective, observational study conducted at Orthopaedic Department of Cochin Hospital, Paris, between 1st January 2010 and 31 December 2015. Patients with a microbiologically proven PJI, treated with one-stage replacement and 6 weeks (+/1week) of antimicrobial therapy were included. Pearson's-χ2 and Wilcoxon tests were used to compare categorical and continuous variables. RESULTS: Fifty patients with periprosthetic joint infections (42 hip, 8 knee PJI) treated with one-stage replacement arthroplasty were included. Median age was 69.3 years (IQR 24.5-97.4). Infections occurred after a mean of 36 months (IQR 1-216). Bone biopsy cultures were positive for Staphylococcus spp. in 29 patients (58%), Cutibacterium acnes in 19 (38%), Gram-negative bacilli in 6 (12%). Polymicrobial infections occurred in 12 (24%). Intravenous antibiotics were administered for a median of 11 days (IQR 4-45) and 46 patients (92%) were switched to an oral therapy. Medium follow-up was of 32 months (IQR 12-101). Overall remission rate was 90%. CONCLUSIONS: A six-week course of antibiotics in knee and hip PJIs treated with one-stage RA has a satisfactory remission rate in this open study.

Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy.

In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

The immobilization of human spermatozoa by STAT3 inhibitory compound V results from an excessive intracellular amount of reactive oxygen species.

We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca(2+) concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species (ROS). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress. To address this question, spermatozoa were incubated with Stattic V in combination with a caspase inhibitor, a proteasome inhibitor or a cell permeant ROS scavenger. Following incubation in different conditions, sperm motility was evaluated by CASA, acrosomal integrity by FITC conjugated Pisum sativum agglutinin (PSA-FITC) labeling, intracellular pH, and mitochondrial superoxide production by flow cytometry using BCECF and MitoSoxRed dye, respectively. Levels of reduced thiols were assessed by iodoacetamidofluorescein staining on total and on sperm surface proteins, and protein tyrosine phosphorylation was evaluated by western blot. The loss in sperm motility induced by Stattic V was associated with a slight intracellular acidification and an important increase in intracellular superoxide anion. Unlike caspase and proteasome inhibitors, low molecular weight thiols, such as N-acetyl-L-cysteine (NAC), prevented Stattic V-induced sperm immobilization and increase responsiveness to acrosome reaction inducers. NAC also efficiently prevented the production of superoxide anion, mitochondrial membrane depolarization, intracellular acidification and the oxidation of protein free thiols caused by Stattic V. These results show that the deleterious effects of Stattic V on sperm functions are caused directly or indirectly by excessive intracellular ROS production without causing sperm apoptosis or necrosis.

Implication of the human Binder of SPerm Homolog 1 (BSPH1) protein in capacitation.

Binder of SPerm (BSP) proteins are a family of proteins expressed exclusively in the male reproductive tract (seminal vesicles or epididymis) of several mammalian species. They are known to promote capacitation, a sperm maturation step essential for fertilization. Our recent studies have shown that in human, the Binder of SPerm Homolog 1 (BSPH1) is expressed solely in epididymal tissues. The goal of the current study was to characterize BSPH1 and evaluate its effect on different sperm functions. A human recombinant BSPH1 (rec-BSPH1) was produced, purified and refolded. Rec-BSPH1 was found to share many characteristics with other members of the BSP superfamily, as it was able to bind gelatin and heparin as well as capacitate sperm. Rec-BSPH1 had no effect on sperm acrosome reaction or any sperm motility parameters. Native BSPH1 was localized on the equatorial segment, post-acrosomal segment and neck of ejaculated human sperm. Rec-BSPH1, following incubation with washed ejaculated human sperm, exhibited binding patterns similar to the native protein. These results show that the human epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and behaves similarly to its murine epididymal orthologue BSPH1. This study of human BSPH1 brings us one step closer to understanding the importance of this protein in male fertility.

IL-1ß/HMGB1 complexes promote The PGE2 biosynthesis pathway in synovial fibroblasts.

PGE2 is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal enzyme of the PGE2 pathway inducible by proinflammatory cytokines. mPGES-1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High-mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro-arthritogenic role in RA. We aimed to investigate the effects of IL-1ß/HMGB1 complexes on mPGES-1 and other enzymes of the PGE2 pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX-2 inhibition and IL-1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL-1ß significantly increased mPGES-1 and COX-2 expressions as well as PGE2 production, as compared to treatment with HMGB1 or IL-1ß alone. Furthermore, NS-398 reduced the production of IL-6 and IL-8, thus indicating that IL-1ß/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL-1RA completely abolished the induced PGE2 and cytokine production, suggesting an effect mediated through IL-1RI. IL-1ß/HMGB1 complexes promote the induction of mPGES-1, COX-2 and PGE2 in SF. The amplification of the PGE2 biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.

Distribution of androgen and oestrogen receptors-α in the seminal vesicle-related spinal neurones in male rats.

The seminal vesicles are male accessory sex glands that contribute much of the seminal fluid volume. Previous studies have suggested that the majority of autonomic innervations to the rat seminal vesicles originate from the bilateral major pelvic ganglia. Many preganglionic autonomic neurones innervating the pelvic ganglion were expressed androgen receptors (AR) or oestrogen receptor (ER)-α immunoreactivity. However, direct neuroanatomic data regarding the distribution of AR and ER-α in seminal vesicle related-spinal neurones are lacking. In the present study, a nonvirulent pseudorabies virus (PRV-152 strain) was used in a retrograde tracing experiment. Four days after PRV injection into the seminal vesicles of male rats, spinal cord sections were prepared. Double- and triple-fluorescence techniques using AR and ER-α with choline acetyltransferase (ChAT) and PRV were used to investigate the AR and ER-α distribution in the seminal vesicles related spinal neurones in male rats. In lamina X, 14% of the PRV-labelled neurones in the L1-L4 segments and 43% in the L5-S1 segments were double-labelled with AR. In the L1-L4 segments, 6% of PRV-labelled neurones and 26% in the L5-S1 segments were double-labelled with ER-α. In the intermedial cell column area, 10% of PRV-labelled neurones in the L1-L4 segments and 47% of PRV-labelled neurones in the L5-S1 segments were double-labelled with AR. Up to 16% of PRV-labelled neurones in the L5-S1 segments were double-labelled with ER-α. No PRV-labelled neurones in the L1-L4 segments were double-labelled with ER-α. However, for the AR and ER-α/PRV/ChAT triple-fluorescence experiments, very few seminal vesicle preganglionic neurones expressed AR or ER-α. Our data suggests that many spinal interneurones but not preganglionic neurones involved in the seminal vesicle control in male rats were double-labelled with AR or ER-α, and they were mainly located at the parasympathetic level in the spinal cord.

Anti-inflammatory cytokine profile in early human tendon repair.

PURPOSE: The aim of this study was to assess inflammation and the presence and relative levels of cytokines, which may be involved in regulating early human Achilles tendon healing. METHODS: Nine patients with acute Achilles tendon rupture were included, operated on and post-operatively immobilized. Two weeks post-operatively, microdialysis of the peritendinous interstitial compartment was performed in the healing and intact contralateral Achilles tendons. Quantification of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70 and IL-17A was accomplished using a cytometric bead array. Prostaglandin (PG) E2 levels were measured by enzyme immunoassay. RESULTS: None of the patients displayed detectable PGE2 levels. Pro-inflammatory cytokines were below detection levels (IFNγ, IL-12, and IL-17) or did not differ between injured and control tendons (IL-1ß and TNF). Notably, IL-6, IL-8 and IL-10 concentrations in the healing Achilles tendon were significantly elevated: 13-fold (p = 0.009), 28-fold (p = 0.02), and 3.7-fold (p = 0.03), respectively. CONCLUSION: At 2 weeks post-human Achilles tendon rupture, healing is characterized by a resolving inflammatory phase and up-regulation of IL-6, IL-8 and IL-10. The absence of inflammation suggests that at this time point, these cytokines may be associated with anti-inflammatory and regenerative effects on the tendon healing process.

Infected total hip arthroplasty revision: one- or two-stage procedure?

INTRODUCTION: Better outcomes have been reported for two-stage total hip arthroplasty (THA) revision for infection. However, one-stage revision arthroplasty remains an attractive alternative option since it requires only one operation. A decision tree has been developed by the authors in order to determine which type of surgical procedure can be performed safely. The goal of this study was to assess this decision tree for THA replacement in the case of a peri-prosthetic infection. HYPOTHESIS: A one-stage procedure may be as successful as a two-stage procedure provided some criteria are fulfilled. METHODS: A prospective study included 84 patients, all diagnosed with infected THA who had prosthesis replacement. A one-stage exchange was performed in 38 cases and a two-stage procedure in 46 cases. A two-stage procedure was decided in the case of important bone loss or unidentified germ. Postoperatively, patients received intravenous antibiotics (six weeks), then oral antibiotics (six weeks). The main evaluation criterion was the rate of infection eradication at 2 years minimal follow-up since surgery. If new infection was suspected, a hip aspiration was performed to determine whether it was non-eradication (same germ) or a new re-infection (other germ), which was not considered as a failure. RESULTS: The initial infection was cured in 83 out of 84 patients (98.8%), 38 (100%) for the one-stage group and 45 (97.8%) for the two-stage group. Three patients were re-infected with different germs in the two-stage group. Eighty out of 84 (95.2%) patients were infection free, all patients (100%) of the one-stage group and 42 patients (91.3%) of two-stage group. DISCUSSION: If some selection criteria were respected, a high success rate in THA replacement for infection may be achieved with a one-stage procedure. It permits to reduce the costs with no loss of chance for the patients. The decision tree was validated. LEVEL OF EVIDENCE: Level III; prospective case control study.

Infertility in a beef bull due to a failure in the capacitation process.

The objective of this case report was to identify the cause of apparent idiopathic infertility in a Red Angus (beef) bull. Semen was collected by electroejaculation and submitted to a series of assays, including evaluation of sperm motility by computer-assisted sperm analysis (CASA), sperm morphology and DNA integrity, semen cryopreservation, AI, IVF, induction of the acrosome reaction, and determination of the level of sperm proteins associated with bull fertility potential. Total (92 ± 2%) and progressive (79 ± 4%) sperm motility; sperm concentration (1647 ± 429 × 10(6) sperm/mL); proportions of morphologically normal sperm (83 ± 6%) and DNA integrity (96 ± 2), and acrosome-intact sperm (64 ± 4%) exceeded minimum acceptable values. Frozen sperm had good total (58.7 ± 6.7%) and progressive (43.9 ± 9.2%) motility immediately after thawing. However, AI of 16 heifers resulted in no pregnancies and blastocyst production rate (following IVF using sperm from this infertile bull) was nearly identical to that produced using dead sperm (a control of parthenogenesis; 2 ± 2 and 2 ± 3%; respectively P < 0.05). Treatment with a calcium ionophore (A23187) failed to induce the acrosome reaction in sperm from the infertile bull (P < 0.05). Evaluation of several proteins associated with the fertility potential of bulls revealed that the level of Binder Sperm Protein-1 (BSP1), known to be associated with the capacitation process, was much greater on sperm from the infertile bull compared to that of his sire. In conclusion, we inferred that the idiopathic infertility in this bull was caused by a failure to complete the capacitation process.

[Fast-tracking and regional anaesthesia: preliminary feasibility study]. / Fast-tracking en anesthésie locorégionale périphérique: étude préliminaire de faisabilité.

OBJECTIVE: The monitoring in the post-anaesthesia care unit (PACU) improves the safety, the comfort and the analgesia of patients. At present, studies suggest the possibility to bypass the PACU according to the principle of fast-tracking (FT). The aim of this study was to evaluate the feasibility and the safety of a simulated protocol of FT after a regional anaesthesia. PATIENTS AND METHODS: Seven hundred patients were prospectively included in this study over a period of 6 months. METHODS: The Withes' scoring system was used for determining when patients could be safely discharged from PACU. We added a variable concerning the monitoring of surgical site. A minimum score of 14 was required on arrival to the PACU to consider a FT. The success rate of blocks, the use of sedation or general anaesthesia were noted. Adverse events were recorded. RESULTS: The success rate of blocks was 93 %. The score was higher than 14 in 98 % of case on arrival to the PACU. Thirteen adverse events were reported before surgery and/or operating room. No adverse events were reported during the stay in the PACU. CONCLUSION: Regional anaesthesia seems to be an appropriate principle to fast-track the PACU. It could be a way to reduce health care costs, and can offer solution for the PACU congestion problem. In France, the fast-tracking is a marginal concept without any support regulatory. An evolution to such a practice could be considered.

The learning process of the hydrolocalization technique performed during ultrasound-guided regional anesthesia.

BACKGROUND: Because poor echogenicity of the needle remains a safety issue, we decided to analyze the learning process of the hydrolocalization technique (Hloc) performed to continuously identify needle-tip anatomical position during many ultrasound-guided regional anesthesia procedures. METHODS: Ten senior anesthesiologists naïve to the Hloc agreed to participate in the study. They were requested to perform 40 out-of-plane (OOP) approach ultrasound-guided axillary blocks (AB) each using the Hloc. The Hloc, which is a needle-tip localization principle, was performed by means of repetitive injections of a small amount of a local anesthetic solution (0.5-1 ml) under an ultrasound beam. Details of the learning process and skill acquisition of the Hloc were derived from the following parameters: the duration of block placement, a measure of the perceived difficulty of needle-tip visualization, a measure of block placement difficulty, and the amount of local anesthetics solution required for the technique. RESULTS: Four hundred ABs were performed. The success rate of an ultrasound-guided AB was 98%. The Hloc was successful in all patients. Skill acquisition over time of the Hloc was associated with a significant reduction of both the duration and the perceived difficulty of ABs placement. Apprenticeship data revealed that 20 blocks were required to successfully place AB within 5 min in most cases using the Hloc. CONCLUSION: The Hloc performed during the OOP approach of ultrasound-guided regional anesthesia is a simple technique with a relatively short learning process feasible for efficient placement of ABs.

Spinal neurons involved in the control of the seminal vesicles: a transsynaptic labeling study using pseudorabies virus in rats.

The seminal vesicles are male accessory sex glands that mainly contribute the seminal fluid of the ejaculate. Previous studies have suggested that seminal vesicles are supplied by both sympathetic and parasympathetic nerves. However, this conclusion was mainly based on studies in pelvic major ganglions and direct neuroanatomical evidence of spinal neurons innervating the seminal vesicles is still lacking. In order to map the spinal nerve circuit innervating the seminal vesicles, the present study used the pseudorabies virus (PRV) retrograde tracing technique in combination with immunohistochemistry. Three groups of rats were prepared: (1) nerves intact; (2) right hypogastric nerve and bilateral pelvic nerves sectioned; (3) right pelvic and bilateral hypogastric nerves sectioned. For the intact group, 3 to 5 days after injection of PRV into the left seminal vesicle in male rats, immunohistochemistry for PRV was performed to map the control circuit. Double immunofluorescence experiments against PRV and choline acetyltransferase (ChAT) were performed to discriminate preganglionic neurons and interneurons. Double detection of PRV and galanin (GAL) was also performed to identify lumbar spinothalamic (LSt) cells. Three days after virus injection, both sympathetic and parasympathetic preganglionic neurons were retrograde-labeled. Four days after injection of PRV into the seminal vesicles, PRV-infected neurons were found in the dorsal horn, ventral horn, dorsal gray commissure (DGC), medial gray matter and intermediolateral cell column (IML) from T13 to S1. For the group with an intact hypogastric nerve, 4 days after injection of PRV into the seminal vesicles, PRV-infected neurons were mainly located in DGC and IML of spinal lumbar segments (L) 1-L2. However, in the group with an intact pelvic nerve, PRV-infected neurons were mainly located in DGC of L5-S1 spinal segments. At the L3-L4 level, most of the virus-labeled neurons around the central canal expressed immunoreactivity for GAL, strongly suggesting that they could be LSt cells. These anatomical data support the idea that the sympathetic and parasympathetic nervous system are both involved in the control of the seminal vesicles and we demonstrated a connection between preganglionic neurons innervating the seminal vesicles and LSt cells which play a crucial role in coordinating the spinal control of ejaculation.

[Ultrasound guided regional anaesthesia: an effective method for cleaning the probes]. / Anesthésie locorégionale périphérique sous échographie: efficacité de stratégie de décontamination de la sonde.

OBJECTIVES: The objective of this study was to determine if the ultrasound probe can act as a vector for cross-infection and to compare two cleaning methods for ultrasound probes in order to limit or control the transmission risk. STUDY DESIGN: Prospective study. PATIENTS AND METHODS: The first part of the study (P1) was conducted to evaluate the possibility of the ultrasound probe to serve as a source of cross-contamination. Thirty blocks were placed under ultrasound guidance in elective outpatients. After each procedure (Proc), the ultrasound probe was decontaminated/cleaned using either an antiseptic solution spray (AS: n=15) or just wiped with two dry paper sheets (DP: n=15), in a randomly assigned order. Bacteriological samples were collected before and after each decontamination/cleaning methods and inoculated on a chocolate agar plates. The second part of the study (P2) was conducted to compare the effectiveness of two cleaning methods for ultrasound probes. The ultrasound probes were exposed to a large inoculum of three bacteria (Inoc). They were then cleaned/decontaminated using either DP (n=10) or AS (n=10), in a randomly assigned order. Bacteriological samples were collected before and after each cleaning/decontamination methods and inoculated on a chocolate agar plates. RESULTS: During P1, after Pro, all probes were found to be sterile before and after both AS and DP. During P2, after Inoc, all probes were found infected (CFU>150) but were considered sterile (CFU<10) after both DP and AS. CONCLUSION: The results of this study suggest that the risk of cross-infection during ultrasound guidance in locoregional anaesthesia is really low. Our data suggest that wiping ultrasound probe with two dry paper sheets is an adequate cleaning method to prevent cross-contamination risk.

Commercial sex and HIV transmission in mature epidemics: a study of five African countries.

The study compares the association between using the services of commercial sex workers and male HIV seroprevalence in five African countries: Ghana, Kenya, Lesotho, Malawi and Rwanda. The HIV seroprevalence among men who 'ever paid for sex' was compared with controls who 'never paid for sex'. Results were based on 12,929 eligible men, aged 15-59 years, interviewed in Demographic and Health Surveys. The odds ratio of HIV seroprevalence associated with ever paying for sex was 1.89 (95% confidence interval = 1.57-2.28), with only minor differences by country. The results were stable in multivariate analysis after controlling for available potential cofactors (data on non-sexual routes of transmission were not available). Given the relatively small proportion of men involved, the risk attributable to 'ever paying for sex' remained low: 7.1% in univariate analysis and 4.4% after adjustment, and it varied among countries (range 1.3-9.4%). These results match previous observations that commercial sex seems to play a minor role in the spread of HIV in mature epidemics.