A hundred-year-old mystery-the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae).

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group's reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).

Vascularization inside the epidermis of Neotropical anurans (Nobleobatrachia).

Anuran skin is a dynamic organ involved in essential functions that strongly correlate with specific morphological traits. Particularly, gas exchange has been associated with epidermal modifications, such as reduced cell layers and increased vascularization. Here, we describe the epidermal morphology and its association with capillary networks in the dorsal skin of 103 Neotropical anurans (Nobleobatrachia) from different ecomorphs and habitats. Additionally, we examined the lateral and ventral skin for a subset of these species. We report intraepidermal capillaries in (i) dorsal skin of Lepidobatrachus laevis and Lepidobatrachus llanensis (burrowing and semi-aquatic Chacoan species), Hyloscirtus colymba and Hyloscirtus palmeri (arboreal species from humid forests), and Alsodes neuquensis and 15 Telmatobius spp. (aquatic and semi-aquatic species from cold environments); (ii) lateral skin of Boana benitezi and H. colymba (arboreal species from humid forests), and (iii) ventral skin of B. benitezi, H. colymba, Atelognathus patagonicus (aquatic species from cold environments), and four Chacoan species, Chacophrys pierottii, Ceratophrys cranwelli (burrowing/terrestrial species), and Lepidobatrachus asper and L. llanensis (burrowing/semi-aquatic species). Also, verrucae hydrophilicae were observed exclusively in the ventral skin of Leptodactylus fuscus, Leptodactylus laticeps (terrestrial and Chacoan species), and B. benitezi. Regardless of the skin region, the capillaries always penetrate the epidermis from the dermis, while epidermal cell layers are flattened. Our findings support previous hypotheses stating that the environment where species occur influences skin changes related to cutaneous respiration (intraepidermal capillaries in different body regions) and water absorption (intraepidermal capillaries associated with verrucae hydrophilicae within ventral skin). Also, phylogeny might influence the development of these structures, as revealed by the presence of intraepidermal capillaries in almost all analyzed species of Telmatobius. Finally, the co-occurrence of verrucae hydrophilicae in the ventral skin of hylids from humid forests, and leptodactylids from the subhumid Chacoan region suggest an independent origin.

Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein.

Applying rational design, we developed 17 kDa cyanobacteriochrome-based near-infrared (NIR-I) fluorescent protein, miRFP718nano. miRFP718nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues. miRFP718nano has maximal emission at 718 nm and an emission tail in the short-wave infrared (SWIR) region, allowing deep-penetrating off-peak fluorescence imaging in vivo. The miRFP718nano structure reveals the molecular basis of its red shift. We demonstrate superiority of miRFP718nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract, mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation.

Glassfrogs conceal blood in their liver to maintain transparency.

Transparency in animals is a complex form of camouflage involving mechanisms that reduce light scattering and absorption throughout the organism. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells (RBCs) that strongly attenuate light. Here, we document how glassfrogs overcome this challenge by concealing these cells from view. Using photoacoustic imaging to track RBCs in vivo, we show that resting glassfrogs increase transparency two- to threefold by removing ~89% of their RBCs from circulation and packing them within their liver. Vertebrate transparency thus requires both see-through tissues and active mechanisms that "clear" respiratory pigments from these tissues. Furthermore, glassfrogs' ability to regulate the location, density, and packing of RBCs without clotting offers insight in metabolic, hemodynamic, and blood-clot research.

High-Frequency 3D Photoacoustic Computed Tomography Using an Optical Microring Resonator.

3D photoacoustic computed tomography (3D-PACT) has made great advances in volumetric imaging of biological tissues, with high spatial-temporal resolutions and large penetration depth. The development of 3D-PACT requires high-performance acoustic sensors with a small size, large detection bandwidth, and high sensitivity. In this work, we present a new high-frequency 3D-PACT system that uses a micro-ring resonator (MRR) as the acoustic sensor. The MRR sensor has a size of 80 µm in diameter, and was fabricated using the nanoimprint lithography technology. Using the MRR sensor, we have developed a transmission-mode 3D-PACT system that has achieved a detection bandwidth of ~23 MHz, an imaging depth of ~8 mm, a lateral resolution of 114 µm, and an axial resolution of 57 µm. We have demonstrated the 3D PACT's performance on in vitro phantoms, ex vivo mouse brain, and in vivo mouse ear and tadpole. The MRR-based 3D-PACT system can be a promising tool for structural, functional, and molecular imaging of biological tissues at depths.

Multiple origins of green coloration in frogs mediated by a novel biliverdin-binding serpin.

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.

Effect of VSL#3 Probiotic in a Patient with Glycogen Storage Disease Type Ia and Irritable Bowel Disease-like Disease.

Gut Inflammatory bowel disease (IBD) is a group of chronic gastrointestinal disorders characterised by relapsing and remitting inflammation of the gastrointestinal tract. The two most common types of IBDs are ulcerative colitis and Crohn's disease. Patients with glycogen storage disease (GSD) type Ia present with gastrointestinal symptoms such as recurrent abdominal pain, bloating and changes in stool form or frequency, which is clinically difficult to distinguish from IBD. We report the case of a 36-year-old man with GSD type Ia and IBD-like disease. A commercial probiotic (VSL#3®) was chosen as a nutritional supplement treatment because of its high content of microbial species and strains. Three different tests were performed: normal-dose, no-dose and half-dose tests. The study periods for the normal-dose, no-dose and half-dose tests were 4 weeks from the treatment initiation, 72 h from the end of the previous period and 4 weeks to 6 months after the end of the 72-h period, respectively. When the probiotic treatment was stopped, he experienced several symptoms similar to those before the start of the treatment. The intestinal symptoms were less severe with the half-dose nutritional supplement treatment than with no treatment. Probiotics may reduce the number of irritable gut episodes and improve the patient's well-being and overall quality of life. More studies are needed to determine whether the improvement in more severe cases of GSD is due mainly to changes in the composition of the gut microbiota, as in this patient.

Ixabepilone and Carboplatin for Hormone Receptor Positive/HER2-neu Negative and Triple Negative Metastatic Breast Cancer.

BACKGROUND: Hormonal therapies and single-agent sequential chemotherapeutic regimens are the standards of care for HER2- metastatic breast cancer (MBC). However, treating patients with hormone-refractory and triple negative (TN) MBC remains challenging. We report the results of combined ixabepilone and carboplatin in a single-arm phase II trial. PATIENTS AND METHODS: In the present prospective analysis of hormone receptor-positive (HR+)/HER2- and TN MBC cohorts, patients could have received 0 to 2 chemotherapy regimens for MBC before enrollment. All patients received ixabepilone 20 mg/m2 and carboplatin (area under the curve, 2.5) on days 1 and 8 every 21 days. The primary endpoint was the objective response rate (ORR). The secondary objectives included progression-free survival (PFS), clinical benefit rate (CBR), overall survival (OS), and toxicity. RESULTS: We enrolled 54 HR+ and 49 TN patients (median, 1 previous chemotherapy regimen for metastatic disease; most in addition to adjuvant chemotherapy). The ORR was 34% and 30.4% for the HR+ and TN patients, respectively, with a corresponding CBR of 56.6% and 41.3%. The ORRs were similar in taxane-pretreated patients (ORR, 31.4% and 28.6% for HR+ and TN patients, respectively). The median OS was 17.9 months for HR+ patients and 12.5 months for TN patients. The median PFS was similar for both groups at 7.6 months. Grade 3/4 nonhematologic toxicities included neuropathy (9%) and fatigue (8%). Nine patients developed grade 3/4 neuropathy, 7 of whom had received previous taxane treatment. CONCLUSION: Ixabepilone plus carboplatin is active even in later-line HR+ and TN disease. Toxicities were manageable without cumulative myelosuppression. This combination is a reasonable option for those patients with MBC who require combination chemotherapy.

Naturally occurring fluorescence in frogs.

Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual perception in terrestrial environments.

[Application of an obesity treatment protocol for 2 years]. / Aplicación de un protocolo de tratamiento de obesidad durante 2 años.

OBJECTIVE: To evaluate the effectiveness of a clinic protocol used in the Complexo Hospitalario Universitario de Vigo (CHUVI) for obese outpatients. PATIENTS AND METHODS: The study included 47 obese outpatients. All of them were evaluated in clinical department and applied the obesity protocol for a period of 2 years. Variables as weight, BMI and levels of obesity in the initial and final time were evaluated. RESULTS AND DISCUSSION: In obese patients between 26 and 65 years was observed a tendency to reduce their degree of obesity, with significant differences in 2012 compared to 2010. There are differences in behavior between men and women in terms of change in obesity graduation. CONCLUSIONS: In our study we found differences in behavior between men and women in terms of grade change in obesity, in women there is a greater tendency to reduce.

Objetivo: Evaluar la eficacia de un protocolo clínico para pacientes obesos utilizado en la consulta de obesidad del Complexo Hospitalario Universitario de Vigo (CHUVI). Pacientes y métodos: En el estudio participaron 47 pacientes procedentes de la consulta de obesidad del CHUVI. Todos ellos fueron evaluados en consulta y siguieron el protocolo de obesidad durante un periodo de 2 años. Se evaluaron variables como el peso, el IMC y los grados de obesidad en el momento inicial y final. Resultados y discusión: En pacientes obesos entre 26 y 65 años se observa una tendencia a disminuir su grado de obesidad, con diferencias significativas en el 2012 respecto al 2010. Hay diferencias de comportamiento entre hombres y mujeres en cuanto al cambio de graduación en la obesidad. Conclusiones: En nuestro estudio hemos comprobado diferencias de comportamiento entre hombres y mujeres en cuanto al cambio de graduación en la obesidad; en las mujeres hay una mayor tendencia a reducirlo.

Aplicación de un protocolo de tratamiento de obesidad durante 2 años / Application of an obesity treatment protocol for 2 years

Objetivo: Evaluar la eficacia de un protocolo clínico para pacientes obesos utilizado en la consulta de obesidad del Complexo Hospitalario Universitario de Vigo (CHUVI). Pacientes y métodos: En el estudio participaron 47 pacientes procedentes de la consulta de obesidad del CHUVI. Todos ellos fueron evaluados en consulta y siguieron el protocolo de obesidad durante un periodo de 2 años. Se evaluaron variables como el peso, el IMC y los grados de obesidad en el momento inicial y final. Resultados y discusión: En pacientes obesos entre 26 y 65 años se observa una tendencia a disminuir su grado de obesidad, con diferencias significativas en el 2012 respecto al 2010. Hay diferencias de comportamiento entre hombres y mujeres en cuanto al cambio de graduación en la obesidad. Conclusiones: En nuestro estudio hemos comprobado diferencias de comportamiento entre hombres y mujeres en cuanto al cambio de graduación en la obesidad; en las mujeres hay una mayor tendencia a reducirlo (AU)

Objective: To evaluate the effectiveness of a clinic protocol used in the Complexo Hospitalario Universitario de Vigo (CHUVI) for obese outpatients. Patients and methods: The study included 47 obese outpatients. All of them were evaluated in clinical department and applied the obesity protocol for a period of 2 years. Variables as weight, BMI and levels of obesity in the initial and final time were evaluated. Results and discussion: In obese patients between 26 and 65 years was observed a tendency to reduce their degree of obesity, with significant differences in 2012 compared to 2010. There are differences in behavior between men and women in terms of change in obesity graduation. Conclusions: In our study we found differences in behavior between men and women in terms of grade change in obesity, in women there is a greater tendency to reduce (AU)

Structural diversity of nuptial pads in Phyllomedusinae (Amphibia: Anura: Hylidae).

We studied the morphological variation of the nuptial pads using light microscopy and scanning electron microscopy (SEM) in 26 species of phyllomedusines (Anura: Hylidae), representing the five currently recognized genera. All phyllomedusines have single nuptial pads with dark colored epidermal projections (EPs). Spine-shaped EPs occur in Cruziohyla calcarifer, Phrynomedusa appendiculata and in one species of Phasmahyla. The other species have roundish EPs. The density of the EPs on the pad is variable. Species in the Phyllomedusa hypochondrialis Group have EPs with a density that varies between 764 ± 58/mm(2) and 923 ± 160/mm(2). In all other studied species (including the Phyllomedusa burmeisteri and Phyllomedusa perinesos groups, Phyllomedusa camba, Phyllomedusa boliviana, Phyllomedusa sauvagii, Phyllomedusa bicolor, and Phyllomedusa tomopterna) the density of EPs varies between 108 ± 20/mm(2) and 552 ± 97/mm(2). Pores were observed with SEM in C. calcarifer, Agalychnis lemur, Agalychnis moreletii, but its presence is confirmed through histological sections on several other species. Its visibility using SEM seems to be related with the level of separation between adjacent EPs. The pores in the four studied species of Agalychnis are shown with SEM and histological sections to have a characteristic epidermal rim, that is absent in the otherphyllomedusines. Unlike most previous reports on breeding glands, those of phyllomedusines are alcian blue positive, indicating the presence of acidic mucosubstances on its secretions.

Proteoliposome derived cochleate as novel adjuvant.

Cochleate structures (CS) consist in a highly stable lipid structures that have been reported to be a good antigen delivery system. The incorporation of pathogen associated molecular pattern (PAMP) from bacterial membranes into CS became in a promising approach to develop adjuvants, particularly mucosal adjuvants. Therefore, we prepare CS from proteoliposome (PL) obtained from Neisseria meningitidis B (PLCS) and evaluated it for its capability to stimulate the immune system as well as the adjuvant activity. The ability of PLCS to induce Thl polarization was also explored. The results and the easy capability for new antigen incorporation on CS support its use as adjuvant for immunization with a large variety of pathogen derived antigens and different routes of immunization.

Immunological evaluation of bacterial derived Cochleate and proteoliposome as mucosal adjuvants.

We evaluated the potential of two bacterial derived compounds, Cochleate and Proteoliposome (PL), administrated to mice by nasal or oral routes on induction of antibody and cytokine responses. Anti PL IgG and IgA responses were measured by ELISA in saliva, sera or vaginal fluids of immunized mice. Productions of gammaIFN and IL-5 were determined in spleen cells of immunized mice following a recall in vitro with Cochleate or PL. Intranasal administration elicited a higher anti PL IgA response in both saliva and vaginal fluids as compared with oral route. Mice immunized with Cochleate or PL via intranasal or oral route-induced anti PL IgG and IgG2a antibody responses in their sera and vaginal fluids. Spleen cells from these immunized mice produced gammaIFN, but not IL-5, after a recall in vitro with Cochleate or PL. These results show that Cochleate and PL are capable of inducing both systemic and mucosal antibody responses as well as a Th1 type of immunity as evidenced by high gammaIFN and IgG2a antibody responses.

Proteliposome-derived Cochleate as an immunomodulator for nasal vaccine.

Proteoliposome (PL) has been recently used as a protective intramuscular (i.m.) anti-meningococcal BC vaccine. It induces a preferential Th 1 type of immune response. Nevertheless, mucosal protection is mainly mediated by IgA antibody response, which is not usually induced by i.m. vaccination route. IgA antibody production needs the stimulation of Th3 subpopulation, which is also related to the induction of small dose tolerance. We hypothesized that PL-derived Cochleate can induce a specific mucosal IgA and systemic IgG antibody responses. We could show that mice immunized with two or three intranasal doses of PL-derived Cochleate developed significantly increased levels of local anti PL IgA and systemic IgG antibody responses. Thus, our results suggest that PL-derived Cochleate can be used as a promising immunomodulator and delivery system for the development of mucosal, particularly nasal vaccines.

Adjuvant properties of lipopolysaccharide from Neisseria meningitidis serogroup B detoxified and conjugated with tetanus toxoid.

We evaluated the adjuvant properties and toxicity of purified Neisseria meningitidis serogroup B lipopolysaccharide (LPS) conjugated with tetanus toxoid (TT) using a new method of conjugation to obtain amine groups in the polysaccharide structure. The endotoxic activity of treated LPS was reduced 2400 times as determined by Limulus amoebocyte assay and no mortality was observed in Balb/c mice inoculated with detoxified LPS versus 100% mortality in native LPS inoculated mice. The conjugated LPS-TT elicited in mice higher anti-TT IgG2a and IgG1 than unconjugated TT. In addition, high levels of anti-LPS IgG and IgG subclasses were detected in sera. These results evidence the adjuvant activity of detoxified LPS and may suggest that the conjugation to TT changes the LPS immune response from thymus-independent to thymus-dependent.

Scale up of proteoliposome derived Cochleate production.

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern.

Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.

Standardization of Neisseria meningitidis serogroup B colorimetric serum bactericidal assay.

The correlate of protection for serogroup B meningococci is not currently known, but for serogroup C it is believed to be the serum bactericidal assay (SBA). The current SBAs are labor intensive and the variations in protocols among different laboratories make interpretation of results difficult. A colorimetric SBA (cSBA), based on the ability of Neisseria meningitidis serogroup B to consume glucose, leading to acid production, was standardized by using group B strain Cu385-83 as the target. The cSBA results were compared to those obtained for a traditional colony-counting microassay (mSBA). Glucose and bromocresol purple pH indicator were added to the medium in order to estimate growth of cSBA target cell survivors through color change. Different variants of the assay parameters were optimized: growth of target cells (Mueller Hinton agar plates), target cell number (100 CFU/per well), and human complement source used at a final concentration of 25%. After the optimization, three other group B strains (H44/76, 490/91, and 511/91) were used as targets for the cSBA. The selection of the assay parameters and the standardization of cSBA were done with 13 sera from vaccinated volunteers. The titers were determined as the higher serum dilution that totally inhibited the bacterial growth marked by the color invariability of the pH indicator. This was detected visually as well as spectrophotometrically and was closely related to a significant difference in the growth of target cell survivors determined using Student's t test. Intralaboratory reproducibility was +/-1 dilution. The correlation between bactericidal median titers and specific immunoglobulin G serum concentration by enzyme immunoassay was high (r = 0.910, P < 0.01). The bactericidal titers generated by the cSBA and the mSBA were nearly identical, and there was a high correlation between the two assays (r = 0.974, P < 0.01). The standardized cSBA allows easy, fast, and efficient evaluation of samples.