Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals.

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.

Role of PumB antitoxin as a transcriptional regulator of the PumAB type-II toxin-antitoxin system and its endoribonuclease activity on the PumA (toxin) transcript.

The PumAB type-II toxin-antitoxin (TA) system is encoded by pumAB genes that are organized into an operon. This system is encoded by the pUM505 plasmid, isolated from a Pseudomonas aeruginosa clinical strain. The pumA gene encodes a putative RelE toxin protein (toxic component), whereas the pumB gene encodes a putative HTH antitoxin protein. The expression of the PumAB system in Escherichia coli confers plasmid stability. In addition, PumA toxin overexpression in P. aeruginosa possesses the capability to increase bacterial virulence, an effect that is neutralized by the PumB antitoxin. The aim of this study was to establish the mechanism of regulation of the PumAB toxin-antitoxin system from pUM505. By an in silico analysis of the putative regulatory elements, we identified two putative internal promoters, PpumB and PpumB-AlgU (in addition to the already reported PpumAB), located upstream of pumB. By RT-qPCR assays, we determined that the pumAB genes are transcribed differentially, in that the mRNA of pumB is more abundant than the pumA transcript. We also observed that pumB could be expressed individually and that its mRNA levels decreased under oxidative stress, during individual expression as well as co-expression of pumAB. However, under stressful conditions, the pumA mRNA levels were not affected. This suggests the negative regulation of pumB by stressful conditions. The PumB purified protein was found to bind to a DNA region located between the PpumAB and the pumA coding region, and PumA participates in PumB binding, suggesting that a PumA-PumB complex co-regulates the transcription of the pumAB operon. Interestingly, the pumA mRNA levels decreased after incubation in vitro with PumB protein. This effect was repressed by ribonuclease inhibitors, suggesting that PumB could function as an RNAse toward the mRNA of the toxin. Taken together, we conclude that the PumAB TA system possesses multiple mechanisms to regulate its expression, as well as that the PumB antitoxin generates a decrease in the mRNA toxin levels, suggesting an RNase function. Our analysis provides new insights into the understanding of the control of TA systems from mobile plasmid-encoded genes from a human pathogen.

[Peri-ictal electrocardiographic alterations in paediatric epilepsy patients. A prospective study]. / Alteraciones electrocardiográficas periictales en pacientes pediátricos con epilepsia. Un estudio prospectivo.

INTRODUCTION: Epileptic seizures can be associated with peri-ictal heart rhythm disorders and even sudden death (SUDEP). Few paediatric studies have been conducted on the subject. AIM: To determine heart rhythm disorders in paediatric patients with epilepsy who were studied with video-electroencephalogram (video-EEG). METHODS: Prospective, observational study in patients under 18 years of age admitted for video-EEG at the Hospital Clinico Red de Salud UC-Christus, Santiago, Chile. A neurological and cardiological evaluation and electrocardiogram (ECG) were performed, in addition to a Holter ECG simultaneously with the video-EEG. RESULTS: Twenty-five patients were studied, 12 with refractory epilepsies, 23/25 focal and 20 on polytherapy. Altogether 768 hours of video-EEG were recorded, including 281 seizures in 15 patients: 157 clinical and 103 generalised. There were no serious arrhythmias, only non-specific or probably benign disorders, mainly incomplete right bundle branch block in 11 patients, which were more frequent in refractory epilepsies (p<0.036). Heart rate variation occurred mainly in seizures >30 seconds. No patient presented SUDEP. CONCLUSIONS: This is the first prospective study, with a new technique, using video-EEG and Holter ECG simultaneously in paediatrics. Despite the high number of seizures and refractory patients, we found no serious heart rhythm disorders, consistent with the clinical evaluation. Right bundle branch block is considered a non-pathological cardiological finding, but was highly prevalent in our sample compared to the normal population, especially in refractory epilepsy. Given that this is a serious event in epilepsy, it is important to further investigate such studies to assess preventive measures.

TITLE: Alteraciones electrocardiográficas periictales en pacientes pediátricos con epilepsia. Un estudio prospectivo.Introducción. Las crisis epilépticas pueden asociarse a alteraciones del ritmo cardíaco periictales e incluso a muerte súbita (SUDEP). Existen escasos estudios pediátricos al respecto. Objetivo. Determinar las alteraciones del ritmo cardíaco en pacientes pediátricos con epilepsia estudiados con videoelectroencefalograma (video-EEG). Métodos. Estudio prospectivo, observacional, en pacientes menores de 18 años ingresados para video-EEG en el Hospital Clínico Red de Salud UC-Christus, Santiago, Chile. Se realizó una evaluación neurológica y cardiológica, electrocardiograma (ECG) y Holter ECG simultáneo al video-EEG. Resultados. Se estudió a 25 pacientes, 12 con epilepsias refractarias, 23/25 focales y 20 en tratamiento con politerapia. Se registraron 768 horas de video-EEG, incluyendo 281 crisis epilépticas en 15 pacientes: 157 clínicas y 103 generalizadas. No hubo arritmias graves; sólo alteraciones inespecíficas o probablemente benignas, principalmente bloqueo incompleto de la rama derecha en 11 pacientes, más frecuente en las epilepsias refractarias (p menor de 0,036). La variación de la frecuencia cardíaca ocurrió principalmente en crisis > 30 segundos. Ningún paciente ha presentado SUDEP. Conclusiones. Éste es el primer estudio prospectivo, con una nueva técnica, usando simultáneamente video-EEG y Holter ECG en pediatría. Pese al alto número de crisis y pacientes refractarios, no encontramos alteraciones graves del ritmo cardíaco, en concordancia con la evaluación clínica. El bloqueo de la rama derecha se considera un hallazgo cardiológico no patológico, pero tuvo una alta prevalencia en nuestra muestra, comparada con la población normal, especialmente en la epilepsia refractaria. Dado que la SUDEP es un evento grave en la epilepsia, es importante ahondar más en este tipo de estudios para evaluar medidas preventivas.

Exploring childhood immunization among undocumented migrants in Sweden - following qualitative study and the World Health Organizations Guide to Tailoring Immunization Programmes (TIP).

OBJECTIVES: National vaccination coverage in Sweden is high. Recurrent outbreaks of measles and rubella however highlight some immunity gaps in the population. Current knowledge about immunization status of undocumented migrant children is scant. The World Health Organization/Europe has developed the Guide to Tailoring Immunization Programmes (TIP) to assist countries in diagnosing barriers and motivators to vaccination in communities with low vaccination coverage. Based on the TIP guide, the objective of this study was to explore determinants to vaccination among undocumented immigrants, using qualitative approach. STUDY DESIGN: The study consisted of three steps: (i) an initial workshop for problem statement; (ii) qualitative research for increased understanding of the vaccination practices of children in the undocumented community; and (iii) a second workshop to incorporate the qualitative interview findings together with data from key stakeholders into a conceptual framework. METHODS: This was a qualitative study featuring interviews of seven undocumented parents recruited at non-governmental clinics, three nurses at Child Health Centers, and information from key stakeholders retrieved at workshops as part of the TIP process. RESULTS: The content analysis revealed two main themes: parental fear of being questioned and parental acceptance of child immunization. Undocumented parents had a positive view and attitude toward childhood immunization but expressed strong fear of being asked for identification papers at healthcare facilities. Owing to lack of knowledge on entitlements of the undocumented among health personnel, parents were incorrectly rejected when seeking care for their children. Frequent mobility among undocumented may limit access to complete the immunization schedule. Undocumented parents mistrust healthcare providers and avoid health facilities, further delaying childrens' access to health care, including immunization services. CONCLUSIONS: The findings of this study confirm the complexity of barriers that undocumented parents face regarding childhood immunization. The TIP guide offers a valuable process for a deeper understanding of the determinants of immunization challenges among undocumented migrants.

Plasmid pUM505 encodes a Toxin-Antitoxin system conferring plasmid stability and increased Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.

Quiste folicular pigmentado perianal: variante múltiple inusual / Perianal pigmented follicular cyst: unusual multiple variant

Los quistes foliculares pigmentados son quistes epiteliales muy infrecuentes. Se describen como lesiones papulares pigmentadas, de carácter benigno, que generalmente se ubican en la cabeza o cuello. Surgen de manera única, siendo la variante múltiple aún menos frecuente. A continuación, se presenta el caso de un hombre joven, con antecedentes de VIH en terapia antiretroviral, quien consultó por la presencia de múltiples lesiones perianales, asintomáticas, de larga data, cuyo resultado histológico resultó compatible con: quistes pigmentados foliculares múltiples.

Pigmented follicular cysts are a very rare epithelial cysts. They are described as a benign papular lesion, that is usually located in the head or neck. They arise as unique, being the multiple variant even less frequent. We present the case of a young man with a history of HIV in antiretroviral therapy, who consulted for the presence of multiple, long-term asymptomatic perianal lesions whose histological result was: compatible with multiple follicular pigmented cysts.

Biosecurity Conditions in Small Commercial Chicken Farms, Bangladesh 2011-2012.

In Bangladesh, highly pathogenic avian influenza H5N1 is endemic in poultry. This study aimed to understand the biosecurity conditions and farmers' perception of avian influenza biosecurity in Bangladeshi small commercial chicken farms. During 2011-2012, we conducted observations, in-depth interviews and group discussions with poultry farmers in 16 farms and in-depth interviews with seven local feed vendors from two districts. None of the farms were completely segregated from people, backyard poultry, other animals, households, other poultry farms or large trees. Wild birds and rodents accessed the farms for poultry feed. Farmers usually did not allow the buyers to bring egg trays inside their sheds. Spraying disinfectant in the shed and removing feces were the only regular cleaning and disinfection activities observed. All farmers sold or used untreated feces as fish feed or fertilizer. Farmers were more concerned about Newcastle disease and infectious bursal disease than about avian influenza. Farmers' understanding about biosecurity and avian influenza was influenced by local vendors. While we seldom observed flock segregation, some farmers used measures that involved additional cost or effort to protect their flocks. These farmers could be motivated by interventions to protect their investment from diseases they consider harmful. Future interventions could explore the feasibility and effectiveness of low-cost alternative biosecurity measures.

Unusually High Mortality in Waterfowl Caused by Highly Pathogenic Avian Influenza A(H5N1) in Bangladesh.

Mortality in ducks and geese caused by highly pathogenic avian influenza A(H5N1) infection had not been previously identified in Bangladesh. In June-July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north-eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza-like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real-time RT-PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small-scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza-like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide appropriate diagnostic testing and detect novel influenza strains.

Genes from pUM505 plasmid contribute to Pseudomonas aeruginosa virulence.

The pUM505 plasmid was isolated from a clinical strain of Pseudomonas aeruginosa. This plasmid contains a genomic island with sequence similar to islands found in chromosomes of virulent P. aeruginosa clinical isolates. The objective of this work was to determine whether pUM505 increases the virulence of P. aeruginosa and to identify the genes responsible for this property. First, using the lettuce-leaf model, we found that pUM505 significantly increases the virulence of P. aeruginosa reference strain PAO1. pUM505 also increased the PAO1 virulence in a murine model and increased cytotoxicity of this strain toward HeLa cells. Thus, we generated a pUM505 gene library of 103 clones in the pUCP20 binary vector. The library was transferred to Escherichia coli TOP10 and P. aeruginosa PAO1 to identify genes. The lettuce-leaf model allowed us to identify three recombinant plasmids that increased the virulence of both E. coli and P. aeruginosa strains. These recombinant plasmids also increased the virulence of the PAO1 strain in mice and induced a cytotoxic effect in HeLa cells. Eleven genes were identified in the virulent transformants. Of these genes, only the pUM505 ORF 2 has homology with a gene previously implicated in virulence. These results indicate that pUM505 contains several genes that encode virulence factors, suggesting that the plasmid may contribute directly to bacterial virulence.

Bioprocessing of plant in vitro systems for the mass production of pharmaceutically important metabolites: paclitaxel and its derivatives.

Taxol (paclitaxel) and its derivatives are microtubule-stabilizing drugs widely used in the treatment of several types of cancer, including mammary, prostate, ovarian and non-small-cell lung carcinoma, as well as AIDS-associated Kaposi's sarcoma and other types of tumor. Taxanes stabilize microtubules by enhancing their polymerization and inhibiting depolymerization. Microtubule dynamics are crucial to mitotic spindle formation and function; therefore, cells exposed to taxanes are unable to undergo chromosomal separation during mitosis, become arrested in the G2/M phases of the cell cycle, and are subsequently targeted for apoptosis. Plant cell cultures are used for industrial-scale biotechnological production of important bioactive plant secondary metabolites, including the anticancer agent paclitaxel. In the last two decades, there have been numerous empirical approaches to improve the biotechnological production of taxanes, leading to the conclusion that treatment of Taxus sp. cells with methyl jasmonate or other elicitors is the most effective strategy. However, little insight has been gained into how the elicitors increase taxane biosynthesis or how this process is regulated. In recent years, with the help of "omics" tools, a rational approach has provided new information about taxane metabolism and its control. Once pathway bottlenecks have been identified, it will be possible to engineer Taxus sp. cell lines with overexpression of genes that control the flux-limiting steps, thus boosting taxane productivity. This review describes the chemical and biological characterization of paclitaxel and its derivatives and discusses future prospects for their biotechnological production.

Metamizol, a non-opioid analgesic, acts via endocannabinoids in the PAG-RVM axis during inflammation in rats.

The most commonly used drugs against pain act by inhibiting the cyclooxygenases (COXs). Metamizol (dipyrone) inhibits the COXs and is widely used in Europe and Latin America as a non-opioid analgesic. One target of metamizol and other non-opioid analgesics is the periaqueductal grey matter (PAG), where they trigger descending inhibition of spinal nociceptive transmission. Also, cannabinoids exert an analgesic action at several structures in the peripheral and central nervous system, including the PAG. The present study investigates whether the antinociceptive action of metamizol in the lateral-ventrolateral (LVL) PAG during inflammation is related to endocannabinoids. In anaesthetized rats, unitary action potentials were recorded from spinal nociceptive neurons with receptive fields in the ipsilateral hind paw. Inflammation of the paw induced neuronal hyperexcitability, which was attenuated by intra-LVL-PAG microinjection of metamizol either at the beginning of inflammation or when hyperexcitability was fully established. In both cases, the antinociceptive effect of metamizol was reduced by a microinjection of AM251, an antagonist at the CB1 cannabinoid receptor, either into the LVL-PAG or into the rostral ventromedial medulla (RVM). The RVM is a downstream structure that funnels PAG-derived descending inhibition into the spinal cord. These results show that endocannabinoids and their CB1 receptor (1) contribute at the LVL-PAG to the antinociceptive effects of metamizol, and possibly other non-opioid analgesics; and (2) participate in the PAG-derived activation of RVM descending antinociceptive influences.

Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.

Changes in partial pressures of respiratory gases during submerged voluntary breath hold across odontocetes: is body mass important?

Odontocetes have an exceptional range in body mass spanning 10(3) kg across species. Because, size influences oxygen utilization and carbon dioxide production rates in mammals, this lineage likely displays an extraordinary variation in oxygen store management compared to other marine mammal groups. To examine this, we measured changes in the partial pressures of respiratory gases ([Formula: see text], [Formula: see text]), pH, and lactate in the blood during voluntary, quiescent, submerged breath holds in Pacific white-sided dolphins (Lagenorhynchus obliquidens), bottlenose dolphins (Tursiops truncatus), and a killer whale (Orcinus orca) representing a mass range of 96-3,850 kg. These measurements provided an empirical determination of the effect of body size on the variability in blood biochemistry during breath hold and experimentally determined aerobic dive limits (ADL) within one taxonomic group (odontocetes). For the species in this study, maximum voluntary breath-hold duration was positively correlated with body mass, ranging from 3.5 min in white-sided dolphins to 13.3 min for the killer whale. Variation in breath-hold duration was associated with differences in the rate of change for [Formula: see text] throughout breath hold; [Formula: see text] decreased twice as fast for the two smaller species (-0.6 mmHg O(2) min(-1)) compared to the largest species (-0.3 mmHg O(2) min(-1)). In contrast, the rate of increase in [Formula: see text] during breath hold was similar across species. These results demonstrate that large body size in odontocetes facilitates increased aerobic breath-hold capacity as mediated by decreased mass-specific metabolic rates (rates of change in [Formula: see text] served as a proxy for oxygen utilization). Indeed the experimentally determined 5 min ADL for bottlenose dolphins was surpassed by the 13.3 min maximum breath hold of the killer whale, which did not end in a rise in lactate. Rather, breath hold ended voluntarily as respiratory gases and pH fell within a narrow range for both large and small species, likely providing cues for ventilation.

Prenatal cocaine exposure increases anxiety, impairs cognitive function and increases dendritic spine density in adult rats: influence of sex.

Cocaine exposure during pregnancy can impact brain development and have long-term behavioral consequences. The present study examined the lasting consequences of prenatal cocaine (PN-COC) exposure on the performance of cognitive tasks and dendritic spine density in adult male and female rats. From gestational day 8 to 20, dams were treated daily with 30 mg/kg (ip) of cocaine HCl or saline. At 62 days of age, offspring were tested consecutively for anxiety, locomotion, visual memory and spatial memory. PN-COC exposure significantly increased anxiety in both sexes. Object recognition (OR) and placement (OP) tasks were used to assess cognitive function. Behavioral tests consisted of an exploration trial (T1) and a recognition trial (T2) that were separated by an inter-trial delay of varying lengths. Male PN-COC subjects displayed significantly less time investigating new objects or object locations during T2 in both OR and OP tasks. By contrast, female PN-COC subjects exhibited impairments only in OR and only at the longest inter-trial delay interval. In addition, gestational cocaine increased dendritic spine density in the prefrontal cortex and nucleus accumbens in both genders, but only females had increased spine density in the CA1 region of the hippocampus. These data reveal that in-utero exposure to cocaine results in enduring alterations in anxiety, cognitive function and spine density in adulthood. Moreover, cognitive deficits were more profound in males than in females.

Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection.

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated as Gram-positive enhancer matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells (DCs) both in vivo and in vitro. These DCs showed increased capacities for secretion of proinflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4(+) and CD8(+) T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life.

Seasonality, estrous cycle characterization, estrus synchronization, semen cryopreservation, and artificial insemination in the Pacific white-sided dolphin (Lagenorhynchus obliquidens).

The reproductive physiology of the Pacific white-sided dolphin, Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%, n=37) and conceptions (83%, n=18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm(2)), and sperm concentration (144.3 x 10(7) sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (n=22) were 31 d long and were comprised of a 10 d follicular and 21 d luteal phase. Ovulation occurred 31.2 h after the onset of the LH surge and 19.3 h after the LH peak. Follicular diameter and circumference within 12 h of ovulation were 1.52 and 4.66 cm respectively. Estrus synchronization attempts with altrenogest resulted in 17 (22%) ovulatory cycles with ovulation occurring 21 d post-altrenogest. Ten AI attempts using cryopreserved semen resulted in five pregnancies (50%). The mean gestation length was 356 days (range 348-367). These data provide new information on the Pacific white-sided dolphin's reproductive physiology and collectively enabled the first application of AI in this species.

Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques.

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.

Changing epidemiology and ecology of highly pathogenic avian H5N1 influenza viruses.

The H5N1 virus currently circulating is continuing to evolve, and it has already resulted in the extension of its host and geographical range. It is likely that H5N1 will become a global problem for the poultry industry. How many of the recent H5N1 changes observed have been induced by changing patterns in poultry raising? A change in attitude on the use of high-quality vaccines is a change that would drastically help in the control of the current epidemic in the poultry industry. This article provides an overview of the changing properties that have been observed during the current H5N1 outbreaks.

Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia?

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.