Protocol to dissect and dissociate the mouse brainstem for single-cell RNA-seq applications.

Processing dissociated cells for transcriptomics is challenging when targeting small brain structures, like brainstem nuclei, where cell yield may be low. Here, we present a protocol for dissecting, dissociating, and cryopreserving mouse brainstem that allows asynchronous sample collection and downstream processing of cells obtained from brainstem tissue in neonatal mice. Although we demonstrate this protocol with the isolated preBötzinger complex and downstream SmartSeq3 cDNA library preparation, it could be readily adapted for other brainstem areas and library preparation approaches.

Prostaglandin E2 Exerts Biphasic Dose Response on the PreBötzinger Complex Respiratory-Related Rhythm.

Inflammation in infants can cause respiratory dysfunction and is potentially life-threatening. Prostaglandin E2 (PGE2) is released during inflammatory events and perturbs breathing behavior in vivo. Here we study the effects of PGE2 on inspiratory motor rhythm generated by the preBötzinger complex (preBötC). We measured the concentration dependence of PGE2 (1 nM-1 µM) on inspiratory-related motor output in rhythmic medullary slice preparations. Low concentrations (1-10 nM) of PGE2 increased the duration of the inspiratory burst period, while higher concentrations (1 µM) decreased the burst period duration. Using specific pharmacology for prostanoid receptors (EP1-4R, FPR, and DP2R), we determined that coactivation of both EP2R and EP3R is necessary for PGE2 to modulate the inspiratory burst period. Additionally, biased activation of EP3 receptors lengthened the duration of the inspiratory burst period, while biased activation of EP2 receptors shortened the burst period. To help delineate which cell populations are affected by exposure to PGE2, we analyzed single-cell RNA-Seq data derived from preBötC cells. Transcripts encoding for EP2R (Ptger2) were differentially expressed in a cluster of excitatory neurons putatively located in the preBötC. A separate cluster of mixed inhibitory neurons differentially expressed EP3R (Ptger3). Our data provide evidence that EP2 and EP3 receptors increase the duration of the inspiratory burst period at 1-10 nM PGE2 and decrease the burst period duration at 1 µM. Further, the biphasic dose response likely results from differences in receptor binding affinity among prostanoid receptors.

Dendritic A-Current in Rhythmically Active PreBötzinger Complex Neurons in Organotypic Cultures from Newborn Mice.

The brainstem preBötzinger complex (preBötC) generates the inspiratory rhythm for breathing. The onset of neural activity that precipitates the inspiratory phase of the respiratory cycle may depend on the activity of type-1 preBötC neurons, which exhibit a transient outward K+ current, IA Inspiratory rhythm generation can be studied ex vivo because the preBötC remains rhythmically active in vitro, both in acute brainstem slices and organotypic cultures. Advantageous optical conditions in organotypic slice cultures from newborn mice of either sex allowed us to investigate how IA impacts Ca2+ transients occurring in the dendrites of rhythmically active type-1 preBötC neurons. The amplitude of dendritic Ca2+ transients evoked via voltage increases originating from the soma significantly increased after an IA antagonist, 4-aminopyridine (4-AP), was applied to the perfusion bath or to local dendritic regions. Similarly, glutamate-evoked postsynaptic depolarizations recorded at the soma increased in amplitude when 4-AP was coapplied with glutamate at distal dendritic locations. We conclude that IA is expressed on type-1 preBötC neuron dendrites. We propose that IA filters synaptic input, shunting sparse excitation, while enabling temporally summated events to pass more readily as a result of IA inactivation. Dendritic IA in rhythmically active preBötC neurons could thus ensure that inspiratory motor activity does not occur until excitatory synaptic drive is synchronized and well coordinated among cellular constituents of the preBötC during inspiratory rhythmogenesis. The biophysical properties of dendritic IA might thus promote robustness and regularity of breathing rhythms.SIGNIFICANCE STATEMENT Brainstem neurons in the preBötC generate the oscillatory activity that underlies breathing. PreBötC neurons express voltage-dependent currents that can influence inspiratory activity, among which is a transient potassium current (IA) previously identified in a rhythmogenic excitatory subset of type-1 preBötC neurons. We sought to determine whether IA is expressed in the dendrites of preBötC. We found that dendrites of type-1 preBötC neurons indeed express IA, which may aid in shunting sparse non-summating synaptic inputs, while enabling strong summating excitatory inputs to readily pass and thus influence somatic membrane potential trajectory. The subcellular distribution of IA in rhythmically active neurons of the preBötC may thus be critical for producing well coordinated ensemble activity during inspiratory burst formation.

Robust Hierarchically Structured Biphasic Ambipolar Oxide Photoelectrodes for Light-Driven Chemical Regulation and Switchable Logic Applications.

Tunable ambipolar photoelectrochemical behavior emerges from microdomains of nanostructured p-type CuFeO2 and n-type Fe2 O3 that arise from a single facile solution-processed thin film. The switchable operation of this system is controlled by chemical, optical, or electronic inputs with a uniquely high photocurrent response (on the order of 1 mA cm-2 ), suitable for robust practical application as an oxygen photoregulator.

Potential market size and impact of hepatitis C treatment in low- and middle-income countries.

The introduction of direct-acting antiviral agents (DAAs) has made hepatitis C infection curable in the vast majority of cases and the elimination of the infection possible. Although initially too costly for large-scale use, recent reductions in DAA prices in some low- and middle-income countries (LaMICs) has improved the prospect of many people having access to these drugs/medications in the future. This article assesses the pricing and financing conditions under which the uptake of DAAs can increase to the point where the elimination of the disease in LaMICs is feasible. A Markov simulation model is used to study the dynamics of the infection with the introduction of treatment over a 10-year period. The impact on HCV-related mortality and HCV incidence is assessed under different financing scenarios assuming that the cost of the drugs is completely paid for out-of-pocket or reduced through either subsidy or drug price decreases. It is also assessed under different diagnostic and service delivery capacity scenarios separately for low-income (LIC), lower-middle-income (LMIC) and upper-middle-income countries (UMIC). Monte Carlo simulations are used for sensitivity analyses. At a price of US$ 1680 per 12-week treatment duration (based on negotiated Egyptian prices for an all oral two-DAA regimen), most of the people infected in LICs and LMICs would have limited access to treatment without subsidy or significant drug price decreases. However, people in UMICs would be able to access it even in the absence of a subsidy. For HCV treatment to have a significant impact on mortality and incidence, a significant scaling-up of diagnostic and service delivery capacity for HCV infection is needed.

Addressing barriers to the prevention, diagnosis and treatment of hepatitis B and C in the face of persisting fiscal constraints in Europe: report from a high level conference.

In the WHO-EURO region, around 28 million people are currently living with chronic viral hepatitis, and 120,000 people die every year because of it. Lack of awareness and understanding combined with the social stigma and discrimination exacerbate barriers related to access to prevention, diagnosis and treatment services for those most in need. In addition, the persisting economic crisis has impacted on public health spending, thus posing challenges on the sustainable investment in promotion, primary and secondary prevention, diagnosis and treatment of viral hepatitis across European countries. The Hepatitis B and C Public Policy Association in cooperation with the Hellenic Center for Disease Prevention and Control together with 10 partner organizations discussed at the Athens High Level Meeting held in June 2014 recent policy developments, persisting and emerging challenges related to the prevention and management of viral hepatitis and the need for a de minimis framework of urgent priorities for action, reflected in a Call to Action (Appendix S1). The discussion confirmed that persisting barriers do not allow the full realisation of the public health potential of diagnosing and preventing hepatitis B and C, treating hepatitis B and curing hepatitis C. Such barriers are related to (a) lack of evidence-based knowledge of hepatitis B and C, (b) limited access to prevention, diagnosis and treatment services with poor patient pathways, (c) declining resources and (d) the presence of social stigma and discrimination. The discussion also confirmed the emerging importance of fiscal constraints on the ability of policymakers to adequately address viral hepatitis challenges, particularly through increasing coverage of newer therapies. In Europe, it is critical that public policy bodies urgently agree on a conceptual framework for addressing the existing and emerging barriers to managing viral hepatitis. Such a framework would ensure all health systems share a common understanding of definitions and indicators and look to integrate their responses to manage policy spillovers in the most cost-effective manner, while forging wide partnerships to sustainably and successfully address viral hepatitis.

Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms.

Study of acute brain stem slice preparations in vitro has advanced our understanding of the cellular and synaptic mechanisms of respiratory rhythm generation, but their inherent limitations preclude long-term manipulation and recording experiments. In the current study, we have developed an organotypic slice culture preparation containing the preBötzinger complex (preBötC), the core inspiratory rhythm generator of the ventrolateral brain stem. We measured bilateral synchronous network oscillations, using calcium-sensitive fluorescent dyes, in both ventrolateral (presumably the preBötC) and dorsomedial regions of slice cultures at 7-43 days in vitro. These calcium oscillations appear to be driven by periodic bursts of inspiratory neuronal activity, because whole cell recordings from ventrolateral neurons in culture revealed inspiratory-like drive potentials, and no oscillatory activity was detected from glial fibrillary associated protein-expressing astrocytes in cultures. Acute slices showed a burst frequency of 10.9 ± 4.2 bursts/min, which was not different from that of brain stem slice cultures (13.7 ± 10.6 bursts/min). However, slice cocultures that include two cerebellar explants placed along the dorsolateral border of the brainstem displayed up to 193% faster burst frequency (22.4 ± 8.3 bursts/min) and higher signal amplitude (340%) compared with acute slices. We conclude that preBötC-containing slice cultures retain inspiratory-like rhythmic function and therefore may facilitate lines of experimentation that involve extended incubation (e.g., genetic transfection or chronic drug exposure) while simultaneously being amenable to imaging and electrophysiology at cellular, synaptic, and network levels.

Where next for hepatitis B and C surveillance?

Hepatitis B and C infections are responsible for significant burden of disease accounting for 1.3 million deaths globally. There is a lack of quality data on the burden of disease due to these infections. One approach to informing policy makers on trends in hepatitis B and C is through case reporting of diagnosed cases. Data on these cases can identify outbreaks of hepatitis and monitor trends in acute and chronic infection. The European Centers for Disease Control (ECDC) has developed standardized case definitions and a harmonized reporting framework. Two articles in this issue summarize the trends in hepatitis B and C infection in Europe. The results show considerable variability in reported cases across countries, reflecting in part differences in testing practices. Risk factor information highlights the continued importance of injecting drug use as a risk factor for hepatitis C infection. Hepatitis case reporting provides valuable information, and more complete reporting will improve the utility of the data. For a comprehensive epidemiologic assessment of the burden of hepatitis, case reporting should be complemented by other sources of data, such as serologic and behavioural surveys.

Challenges towards Economic Fuel Generation from Renewable Electricity: The Need for Efficient Electro-Catalysis.

Utilizing renewable sources of energy is very attractive to provide the growing population on earth in the future but demands the development of efficient storage to mitigate their intermittent nature. Chemical storage, with energy stored in the bonds of chemical compounds such as hydrogen or carbon-containing molecules, is promising as these energy vectors can be reserved and transported easily. In this review, we aim to present the advantages and drawbacks of the main water electrolysis technologies available today: alkaline and PEM electrolysis. The choice of electrode materials for utilization in very basic and very acid conditions is discussed, with specific focus on anodes for the oxygen evolution reaction, considered as the most demanding and energy consuming reaction in an electrolyzer. State-of-the-art performance of materials academically developed for two alternative technologies: electrolysis in neutral or seawater, and the direct electrochemical conversion from solar to hydrogen are also introduced.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

[Aetiologies of pleural effusions in HIV-infected patients in Abidjan, Côte-d'Ivoire]. / Etiologies des pleurésies chez les patients infectés par le VIH a Abidjan, Côte-d'Ivoire.

From December 1995 to March 1996 a cross sectional study was carried out in the pulmonary Medicine Unit of Treichville in Abidjan. In order to specify the main aetiologies of pleural effusion, an investigation was conducted among 35 adult patients (19 men and 16 women) suffering from pleuritis. Overall, the mean age was 32.2 years (range: 19-53 years). All the patients underwent a standard chest x-ray a skin test with 10 units of tuberculin, a whole blood cells count with CD4 T cells count and HIV test. The following analysis were performed on the pleural fluid for all patients: cytological, bacteriological and mycobacteriological examination. Some patients underwent as well a pleural biopsy performed by Abram's needle. Pleural fluid was clear in 24 cases (69%). Empyema was found in 8 cases (23%) and hemorrhagic fluid in 3 cases (9%). Tuberculosis was the dominant aetiology of pleuritis noted in 29 patients (83%), followed by far by non-tuberculous bacterial infections in 6 patients (17%). Tuberculosis associated with common bacterial infections was noted in 3 cases.

Sociodemographic factors associated with participation by HIV-1-positive pregnant women in an intervention to prevent mother-to-child transmission of HIV in Cote d'Ivoire.

Many HIV-1-seropositive women in Africa who are offered antiretroviral prophylaxis to prevent mother-to-child transmission (MTCT) of HIV do not begin interventions. Research on barriers to participation has not addressed the possible effects of women's sociocultural and economic circumstances. We examined these factors at an MTCT prevention programme in Abidjan, Cote d'Ivoire. We interviewed two groups of women after they had received HIV-positive test results and had been invited by the programme staff to return for monthly follow-up visits before beginning short-course zidovudine prophylaxis. Participants (n = 30) completed follow-up visits and prophylaxis. Non-participants (n = 27) refused or discontinued follow-up visits and did not begin zidovudine. Fewer non-participants had been born in Cote d'Ivoire (67% vs. 97%) or were Ivorian nationals (48% vs. 77%); they had lived in the country for less time (21 vs. 26 median years). They were less likely to be French-literate (37% vs. 77%), and more of them reported having had Koranic education only (18% vs. 0). They more often reported miscarriages, stillbirths, or infant deaths (69% vs. 33%), and had partners with low-ranked jobs (63% vs. 30%). Our findings suggest that the non-participants were more marginal socioculturally and economically in Ivorian society than participants. Greater attention to mitigating the effects of broader structural factors on women's participation in interventions may increase the effectiveness of MTCT prevention in Africa.

Effect of an HIV counseling and testing program on AIDS-related knowledge and practices in tuberculosis clinics in Abidjan, Côte d'Ivoire.

SETTING: Two out-patient tuberculosis treatment centers, Abidjan, Côte d'Ivoire. OBJECTIVE: To assess the effect of a human immunodeficiency virus (HIV) counseling and testing program on acquired immune-deficiency syndrome (AIDS) related knowledge and behaviors among persons with newly diagnosed tuberculosis. DESIGN: Since 1994, patients with newly diagnosed tuberculosis have received individual or group HIV pretest counseling, informed consent, free HIV testing for those who consent, and post test counseling. From January 1995 through August 1996 in Abidjan's two largest tuberculosis clinics, knowledge and beliefs about HIV/AIDS were assessed before and immediately after the group pretest sessions, and again 4 months later. RESULTS: Prior to pretest counseling, 68.9% and 68.0% of the 559 enrolled subjects could correctly identify five modes of HIV transmission and five modes of HIV prevention. These proportions increased significantly immediately after pretest counseling (90.0%, 86.6%, respectively), and remained higher 4 months later (83.7%, 87.7%) (all P < 0.01). Among men, consistent condom use during the preceding 4 months with a partner who was not a commercial sex worker increased from 9.9% at enrollment to 23.6% at the 4-month visit (P = 0.001), but not for women (6.3% vs. 9.5%, P = 0.40). CONCLUSIONS: An HIV pretest counseling program conducted in an out-patient tuberculosis clinic was well accepted, and significantly increased the level of HIV/AIDS knowledge and, among men, self-reported condom use.

Cervicovaginal secretory antibodies to human immunodeficiency virus type 1 (HIV-1) that block viral transcytosis through tight epithelial barriers in highly exposed HIV-1-seronegative African women.

Antibodies to human immunodeficiency virus (HIV) of the IgA, IgG, and IgM isotypes and high levels of the HIV suppressive beta-chemokine RANTES (regulated on activation, normally T cell expressed and secreted) were found in the cervicovaginal secretions (CVSs) of 7.5% of 342 multiply and repeatedly exposed African HIV-seronegative female sex workers. The antibodies are part of a local compartmentalized secretory immune response to HIV, since they are present in vaginal fluids that are free of contaminating semen. Cervicovaginal antibodies showed a reproducible pattern of reactivity restricted to gp160 and p24. Locally produced anti-env antibodies exhibit reactivity toward the neutralizing ELDKWA epitope of gp41. Study results show that antibodies purified from CVSs block the transcytosis of cell-associated HIV through a tight epithelial monolayer in vitro. These findings suggest that genital resistance to HIV may involve HIV-specific cervicovaginal antibody responses in a minority of highly exposed HIV-seronegative women in association with other protecting factors, such as local production of HIV-suppressive chemokines.

Is drinking water in Abidjan, Côte d'Ivoire, safe for infant formula?

OBJECTIVE: To survey knowledge, attitudes, and practices regarding water use and infant feeding in the Koumassi District of Abidjan, Côte d'Ivoire, and to evaluate the microbiologic quality of source and stored drinking water. DESIGN: Random-cluster household survey. METHODS: We randomly selected 20 clusters, each comprising six households with at least 1 child aged < or =3 years. In each household, we administered a questionnaire and collected source and stored drinking water samples and tested these for chlorine levels and for total coliform and fecal bacteria count ( Escherichia coli ). RESULTS: Municipal water was used for drinking in 112 (93%) of 120 households, and in 99 (83%), it was stored for later use. By 1 month of age, 97 (90%) of 108 infants given drinking water were given stored water for drinking. In 8 (66%) of 12 households where children were receiving artificial feeding, formula was prepared from municipal water without additional treatment. Stored water had lower levels of free chlorine than source water (median of 0.05 versus 0.2 mg/dl; p <.001), and E. coli was detected in 36 (41%) of 87 stored water samples and 1 (1%) of 108 source water samples ( p <.001). CONCLUSIONS: In the Koumassi District of Abidjan, where municipal water is widely available and of good quality, drinking water is stored in most households, is often contaminated with E. coli, and is given to children at a young age. If replacement feeding is to be more widely used to prevent postnatal transmission of HIV-1, communities using stored water need interventions to make stored water safer.

Lack of induction of interleukin-2-receptor-alpha in patients with tuberculosis and human immunodeficiency virus co-infection: implications for pathogenesis.

Since expression of both interleukin-2 (IL-2) and IL-2-receptor-alpha (IL-2R-alpha) by lymphocytes is inhibited by human immunodeficiency virus (HIV) in vitro, we hypothesized that HIV-co-infection among persons with tuberculosis (TB) might impair T-lymphocyte responses to TB via this mechanism. We measured soluble IL-2R-alpha (sIL-2R-alpha), a surrogate marker of T-lymphocyte activation and proliferation, and soluble tumour necrosis factor receptor I (sTNF-RI) in sera from West African patients categorized into 4 groups: those with TB alone (TB+ HIV-, n = 55), CD4-matched groups with TB and HIV co-infection (TB+ HIV+, n = 50) or HIV infection alone (TB- HIV+, n = 35), and patients with neither disease (TB- HIV-, n = 35). The median level of sIL-2R-alpha was markedly greater in the TB+ HIV- group (1580 U/mL) compared to the TB- HIV- (670 U/mL; P < 0.001) and TB- HIV+ (880 U/mL; P < 0.01) groups. More importantly, the median concentration of sIL-2R-alpha was much lower in the TB+ HIV+ group (855 U/mL) compared to the TB+ HIV- group (1580 U/mL; P < 0.01) despite similar levels of sTNF-RI. These results suggest that T-lymphocyte activation in TB patients is impaired by HIV co-infection and, furthermore, this suppressive effect was independent of numerical depletion of CD4 lymphocytes. Impairment to IL-2-signalling might contribute to the profound impact that HIV has had on both the incidence and the clinicopathological manifestations of TB.

Where do sex workers go for health care? A community based study in Abidjan, Côte d'Ivoire.

OBJECTIVES: To describe health seeking behaviour of female sex workers in Abidjan, Côte d'Ivoire. METHODS: A population based survey among a representative sample of 500 female sex workers and six focus group discussions. RESULTS: The sites of first encounter for care for the last STI episode included a public hospital or health centre (28%), a private clinic (16%), a confidential clinic (13%), a pharmacy (13%), and the informal sector (23%). The agreement between preferred and actual services used was weak (kappa 0.16). CONCLUSIONS: Sex workers expressed interest in seeking STI care in a wide range of public and private healthcare facilities. Those services should be upgraded to better respond to their sexual health needs.

Effect of interventions to control sexually transmitted disease on the incidence of HIV infection in female sex workers.

OBJECTIVE: To compare the seroincidence of HIV infection among female sex workers in Abidjan, Côte d'Ivoire before and during an intervention study to control sexually transmitted diseases (STD) and to study the effect of two STD diagnosis and treatment strategies on the prevalence of STD and on the seroincidence of HIV infection. METHOD: A screening facility for STD and HIV had been available since October 1992 for female sex workers. From June 1994, women who were HIV seronegative or HIV-2 positive during the screening could enroll in the intervention study in which participants reported once a month to a confidential clinic where they received health education, condoms and STD treatment if indicated. Women in the study were randomized either to a basic STD diagnosis and treatment strategy, which included a gynecologic examination when symptomatic, or to an intensive strategy that included a gynecologic examination regardless of symptoms. An outcome assessment every 6 months included a gynecologic examination, HIV serology and laboratory tests for STD. RESULTS: Of 542 women enrolled in the study, 225 (42%) had at least one outcome assessment. The HIV-1 seroincidence rate during the intervention study was significantly lower than before the study (6.5 versus 16.3 per 100 person-years; P = 0.02). During the study, the HIV-1 seroincidence rate was slightly lower in the intensive than in the basic strategy (5.3 versus 7.6 per 100 person-years; P = 0.5). CONCLUSION: National AIDS control programs should consider adopting as policy the type of integrated approach used in this intervention study for HIV prevention in female sex workers.

Lack of effect of chemokine receptor CCR2b gene polymorphism (64I) on HIV-1 plasma RNA viral load and immune activation among HIV-1 seropositive female workers in Abidjan, Côte d'Ivoire.

The prevalence of the CCR2b-V64I mutation among human immunodeficiency virus (HIV)-seropositive and -seronegative female workers and the potential effect of heterozygosity of this mutation on HIV-1 plasma RNA viral load and markers of immune activation were assessed. CCR2b-V64I was detected by polymerase chain reaction, followed by restriction enzymes analysis; plasma viral load was measured by the Amplicor HIV-1 monitor assay and CD4(+) T-cell counts and markers of immune activation by standard three-color FACscan flow cytometry. Of the 260 female workers, 56 (21.5%) were heterozygous for CCR2b-V64I, and 8 (3%) were homozygous. Of the 99 HIV-seronegative female workers, 19 (19.2%) were heterozygous for the CCR2b-V64I mutation compared with 37 (23%) of the 161 HIV-seropositive FSW (P = 0.47). In a univariate analysis of viral load among HIV-seropositive FSW, no difference was noted between those heterozygous for or without the mutation; both groups had plasma viral loads of 5.0 log(10) copies/ml. After controlling for the effects of CD4(+) T-cell counts in a multivariate analysis, no significant difference was observed between the groups in viral load or in markers of immune activation. The data suggest that the presence of the CCR2b mutation has no effect on HIV-1 plasma viral load and markers of immune activation in our study population. The finding that the frequency of this mutation is similar in HIV-seropositive and -seronegative female workers suggests that its presence is not associated with increased risk of HIV infection.