Dietary lutein supplementation improves immunity and antioxidant status of captive Indian leopards (Panthera fusca).

Indian leopards kept in zoos are fed solely on carabeef on bone (CBB) diets. Carabeef contains lesser or no carotenoids. Hence, the captive Indian leopard diets are suspected to be deficient in carotenoids while their wild counterparts acquire these pigments from their natural prey. Lutein is a vital carotenoid that plays its role as an antioxidant and immunomodulator. This experiment investigates the effect of lutein supplementation on antioxidant status, immunity, and stress in captive Panthera fusca fed CBB diets. Nine leopards were used based on 3 × 3 replicated Latin square designs in the experiment. Groups CON, LUT20, and LUT40 were supplemented with 0, 20, and 40 ppm of lutein, respectively. Each experiment comprised of 10 days of wash-out period, 11 days of adaptation, and 4 days of collection. Digestibility of crude protein (CP) was higher (p < .01) in groups LUT20 and LUT40. Serum concentration of protein, globulin, urea (p < .05), total carotenoids, total antioxidant capacity (TAC), catalase (CAT) activity, and lymphocyte transformation test (LTT) index were higher (p < .001) in groups LUT20 and LUT40. Activity of superoxide dismutase (SOD) and serum concentration of immunoglobulin were higher (p < .001) in group LUT20. Serum concentration of malonaldehyde (MDA) and fecal concentration of cortisol decreased (p < .001) in groups LUT20 and LUT40. Serum concentration of total immunoglobulin (µg/ml) and LTT were higher in group LUT20. Fecal concentration of cortisol (ng/g) was lower in LUT20 and LUT40. The study concludes that supplementation of lutein at 20 ppm would improve antioxidant status and immunity and alleviate stress in captive Indian leopards.

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.

Tracking neural crest cell cycle progression in vivo.

Analysis of cell cycle entry/exit and progression can provide fundamental insights into stem cell propagation, maintenance, and differentiation. The neural crest is a unique stem cell population in vertebrate embryos that undergoes long-distance collective migration and differentiation into a wide variety of derivatives. Using traditional techniques such as immunohistochemistry to track cell cycle changes in such a dynamic population is challenging, as static time points provide an incomplete spatiotemporal picture. In contrast, the fluorescent, ubiquitination-based cell cycle indicator (Fucci) system provides in vivo readouts of cell cycle progression and has been previously adapted for use in zebrafish. The most commonly used Fucci systems are ubiquitously expressed, making tracking of a specific cell population challenging. Therefore, we generated a transgenic zebrafish line, Tg(-4.9sox10:mAG-gmnn(1/100)-2A-mCherry-cdt1(1/190)), in which the Fucci system is specifically expressed in delaminating and migrating neural crest cells. Here, we demonstrate validation of this new tool and its use in live high-resolution tracking of cell cycle progression in the neural crest and derivative populations.

Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Rapid adaptive optical recovery of optimal resolution over large volumes.

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

Sensational placodes: neurogenesis in the otic and olfactory systems.

For both the intricate morphogenetic layout of the sensory cells in the ear and the elegantly radial arrangement of the sensory neurons in the nose, numerous signaling molecules and genetic determinants are required in concert to generate these specialized neuronal populations that help connect us to our environment. In this review, we outline many of the proteins and pathways that play essential roles in the differentiation of otic and olfactory neurons and their integration into their non-neuronal support structures. In both cases, well-known signaling pathways together with region-specific factors transform thickened ectodermal placodes into complex sense organs containing numerous, diverse neuronal subtypes. Olfactory and otic placodes, in combination with migratory neural crest stem cells, generate highly specialized subtypes of neuronal cells that sense sound, position and movement in space, odors and pheromones throughout our lives.

A novel HoxB cluster protein expressed in the hindbrain and pharyngeal arches.

We describe a novel zebrafish line that fluorescently tags a previously unknown protein, CT74a, allowing us to follow its endogenous expression in real time and at subcellular resolution in live embryos. Our results showed that CT74a-Citrine fusion protein is expressed in the developing pharyngeal arches, hindbrain, and fin buds in a pattern highly reminiscent of transcription factors belonging to anterior Hox gene families, including expression in a subset of neuronal nuclei. Consistent with this, splinkerette-PCR revealed that CT74a-Citrine's genomic integration is within the HoxB region, and 3' RACE demonstrated that its downstream coding sequence has no recognizable homology. Thus, CT74a is a previously unknown protein located within the HoxB cluster adjacent to Hoxb4a and is expressed in a Hoxb4a-like pattern.

Crystal lattice determination of ZnSe nanowires with polarization-dependent second harmonic generation microscopy.

We demonstrate a noninvasive optical microscopy technique based on polarization-dependent second harmonic generation for determining the crystal lattice structure and microscopic heterogeneities within individual nanostructures. Differentiation between periodically twinned and wurtzite ZnSe nanowires (NWs) was demonstrated, and measurement of the cubic lattice rotation orientation around the NW axis was determined within 1° accuracy. Zinc blende NWs were differentiated from wurtzite. The technique can be used for quality inspection and optimization of growth conditions for nanostructures.

Optimization of Newtonian fluid pressure in microcantilever integrated flexible microfluidic channel for healthcare application.

The demand for microfluidic pressure sensors is ever-increasing in various industries due to their crucial role in controlling fluid pressure within microchannels. While syringe pump setups have been traditionally used to regulate fluid pressure in microfluidic devices, they often result in larger setups that increase the cost of the device. To address this challenge and miniaturize the syringe pump setup, the researcher introduced integrated T-microcantilever-based microfluidic devices. In these devices, microcantilevers are incorporated, and their deflections correlate with the microchannel's pressure. When the relative pressure of fluid (plasma) changes, the T-microcantilever deflects, and the extent of this deflection provides information on fluid pressure within the microchannel. In this work, finite element method (FEM) based simulation was carried out to investigate the role of material, and geometric parameters of the cantilever, and the fluid viscosity on the pressure sensing capability of the T-microcantilever integrated microfluidic channel. The T-microcantilever achieves a maximum deflection of 127µm at a 5000µm/s velocity for Young's modulus(E) of 360 kPa of PDMS by employing a hinged structure. On the other hand, a minimum deflection of 4.05 × 10-5µm was attained at 5000µm/s for Young's modulus of 1 TPa for silicon. The maximum deflected angle of the T-cantilever is 20.46° for a 360 kPa Young's modulus while the minimum deflection angle of the T-cantilever is measured at 13.77° for 900 KPa at a fluid velocity of 5000µm s-1. The T-cantilever functions as a built-in microchannel that gauges the fluid pressure within the microchannel. The peak pressure, set at 8.86 Pa on the surface of the cantilever leads to a maximum deflection of 0.096µm (approximately 1µm) in the T-cantilever at a 1:1 velocity ratio. An optimized microfluidic device embedded with microchannels can optimize fluid pressure in a microchannel support cell separation.

Unambiguous identification of recombination lines in single zinc-blende ZnSe nanowires in direct relation to their microstructure.

We present results on the low-temperature photoluminescence characterization of individual ZnSe nanowires, whose crystal structure was determined to be zinc-blende by transmission electron microscopy on the same individual nanowires as were studied optically. The photoluminescence response from single ZnSe nanowires was found to be dominated by excitonic emission due to native point defects, while no emission peaks related to the unintentional impurities were detected. Two strong photoluminescence lines were observed at 2.785 eV and 2.780 eV, assigned to the excitons bound to deep neutral acceptors related to the vacancies of Zn (V(Zn)) and complexes of V(Zn), respectively. Another recombination peak at 2.800 eV related to the free exciton emission in ZnSe was also observed. Longitudinal optical-phonon replicas of up to three orders were seen for both lines, and the average number of emitted phonons was also determined. The excitonic emission linewidths of 1.5 meV were observed from individual nanowires, which are the narrowest excitonic linewidths reported so far for ZnSe nanowires. The optical response from a single nanowire was also compared to that from a bundle of nanowires, and it was found that the linewidths of excitonic emission from the bundle of nanowires were slightly larger than those from single nanowires, due to the effects of ensemble broadening. It is also suggested that in the case of a bundle of nanowires, the broadening is limited by the nanowire which exhibits the largest excitonic linewidth.

miRNA-processing enzyme Dicer is necessary for cardiac outflow tract alignment and chamber septation.

MicroRNAs (miRNAs) have previously been implicated in a number of developmental processes, including development of the ventricular myocardium of the heart. To determine what, if any, additional roles miRNAs play in cardiogenesis, we deleted the miRNA-processing enzyme Dicer specifically in the developing murine heart. Embryos lacking cardiac Dicer lived longer than reported in previous studies using different alleles to remove cardiac Dicer activity and displayed a highly penetrant phenotype of double outlet right ventricle with a concurrent ventricular septal defect. Before the defect's onset, Pitx2c and Sema3c, both required for outflow tract morphogenesis, were up-regulated in Dicer-deficient hearts. Interestingly, mesenchymal apoptosis in the outflow tract normally required for outflow tract alignment was greatly decreased in the mutants, likely contributing directly to the observed phenotype. In sum, we demonstrate here a specific developmental process, that of outflow tract morphogenesis, being hindered by the deletion of miRNAs during cardiogenesis.

Injection of human neuroblastoma cells into neural crest streams in live zebrafish embryos.

Cancer cell behavior is highly microenvironment dependent, but we have a limited understanding of malignant cell-microenvironment interactions in vivo. Here, we describe a protocol for xenotransplanting human neuroblastoma (NB) cells into streams of migrating neural crest stem cells in zebrafish embryos, followed by confocal time-lapse imaging and cell tracking. This high-resolution model system facilitates the quantitative spatiotemporal analysis of cancer cell-cell and cell-environment interactions. For complete details on the use and execution of this protocol, please refer to Treffy et al. (2021).

Machine Learning Technique to Detect and Classify Mental Illness on Social Media Using Lexicon-Based Recommender System.

The emergence of social media has allowed people to express their feelings on products, services, films, and so on. The feeling is the user's view or attitude towards any topic, object, event, or service. Overall, feelings have always influenced people's decision-making. In recent years, emotions have been analyzed intensively in natural language, but many problems still have to be watched. One of the most important problems is the lack of precise classification resources. Most of the research into feeling gradation is concerned with the issue of polarity grading, although, in many practical applications, this relatively grounded feeling measure is insufficient. Design methods are therefore essential, which can accurately classify feelings into a natural language. The principal goal of the research is to develop an overflow of grammatical rules-based classification of Indian language tweets. In this work, three main challenges are identified to classify feelings in Indian language tweets and possible methods for tackling such issues. Firstly, it has been found that the informal nature of tweets is crucial for the classification of feelings. Based on the tweets, the mental illness of the person has been classified. Therefore, to categorize Indian language tweets, a combination of grammar rules based on adjectives and negations is proposed. Secondly, people often express their feelings with slang words, abbreviations, and mixed words. A technique called field tags is used to include nongrammatical arguments such as slang words and diverse words. Thirdly, if a tweet is more complex, the morphological richness of the Indian language results in a loss of performance. The grammar rules are embedded in N-gram techniques and machine learning methods. These methods are grouped into three approaches, which functionally predict Indian language tweets with syntactic words.

A Liver Damage Prediction Using Partial Differential Segmentation with Improved Convolutional Neural Network.

Background: The liver is one of the most significant and most essential organs in the human body. It is divided into two granular lobes, one on the right and one on the left, connected by a bile duct. The liver is essential in the removal of waste products from human food consumption, the creation of bile, the regulation of metabolic activities, the cleaning of the blood by sensitizing digestive management, and the storage of vitamins and minerals. To perform the classification of liver illnesses using computed tomography (CT scans), two critical phases must first be completed: liver segmentation and categorization. The most difficult challenge in categorizing liver disease is distinguishing the liver from the other organs near it. Methodology. Liver biopsy is a kind of invasive diagnostic procedure, widely regarded as the gold standard for accurately estimating the severity of liver disease. Noninvasive approaches for examining liver illnesses, such as blood serum markers and medical imaging (ultrasound, magnetic resonance MR, and CT) have also been developed. This approach uses the Partial Differential Technique (PDT) to separate the liver from the other organs and Level Set Methodology (LSM) for separating the cancer location from the surrounding tissue based on the projected pictures used as input. With the help of an Improved Convolutional Classifier, the categorization of different phases may be accomplished. Results: Several accuracies, sensitivity, and specificity measurements are produced to assess the categorization of LSM using an Improved Convolutional classifier. Approximately, 97.5% of the performance accuracy of the liver categorization is achieved with a 94.5% continuous interval (CI) of [0.6775 1.0000] and an error rate of 2.1%. The suggested method's performance is compared to that of two existing algorithms, and the sensitivity and specificity provide an overall average of 96% and 93%, respectively, with 95% Continuous Interval of [0.7513 1.0000] and [0.7126 1.0000] for sensitivity and specificity, respectively.

Non-cardiac Manifestations in Adult Patients With Mucopolysaccharidosis.

Mucopolysaccharidoses (MPS) are a heterogeneous group of disorders that results in the absence or deficiency of lysosomal enzymes, leading to an inappropriate storage of glycosaminoglycans (GAGs) in various tissues of the body such as bones, cartilage, heart valves, arteries, upper airways, cornea, teeth, liver and nervous system. Clinical manifestations can become progressively exacerbated with age and affect their quality of life. Developments in advanced supportive treatment options such as enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT) may have improved patients' life span. Adult MPS patients require specialist clinical surveillance long-term. In many cases, in addition to the MPS-related health problems, they may develop age-related complications. Considering the complexity of their clinical manifestations and lack of guidelines on the management of adult MPS disorders, multispecialty and multidisciplinary teams' care is essential to diagnose and treat health problems that are likely to be encountered. This review presents non-cardiac clinical manifestations, their pathophysiology, management and long-term outcomes in adult MPS patients.

Rabies DNA vaccine encoding lysosome-targeted glycoprotein supplemented with Emulsigen-D confers complete protection in preexposure and postexposure studies in BALB/c mice.

The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for an improved rabies vaccine. Thus, DNA vaccine based on lysosome-targeted glycoprotein of the rabies virus was evaluated in BALB/c mice. It imparted partial protection (60%) against challenge with 20 LD(50) of the challenge virus standard (CVS) strain of rabies virus. To improve the outcome of vaccination, to ultimately enhance the immune response, we investigated different routes for DNA vaccine delivery, varied doses of DNA, and the influence of adjuvant supplementation. The highest immune response pertaining to IgG antibody titer, with a predominantly IgG1/IgG2a subclass distribution, effective cellular immunity, and a high level of rabies virus neutralizing antibodies (RVNAs) was attained by the optimized DNA vaccine formulation comprising intramuscular administration of 100 microg of DNA vaccine supplemented with Emulsigen-D. In preexposure prophylaxis, a 3-dose regimen of this formulation generated a high RVNA titer (32 IU/ml) and conferred complete protection against challenge with 20 LD(50) of CVS. For postexposure efficacy analysis, rabies was experimentally induced with 50 LD(50) of CVS. Subsequent therapy with 5 doses of the formulation completely prevented rabies in BALB/c mice, which maintained protective RVNA titers of 4 IU/ml. The World Health Organization recommended rabies protective titer threshold is 0.5 IU/ml. Thus, this optimized DNA vaccine formulation provides an avenue for preventing and controlling rabies.

Health-seeking behavior and the initial economic impact of patients with open globe injuries seeking treatment in a tertiary care center in India.

PURPOSE: The aim of this study was to assess the health-seeking behavior and the costs of primary treatment in the immediate aftermath of open globe trauma among patients seeking care at a tertiary eye care center in South India. METHODS: This prospective observational study was carried out from June to December 2019. Details of the patient's demographic profile, health-seeking behavior immediately following the trauma and total cost incurred till the completion of primary treatment were collected. RESULTS: Eighty-five patients were recruited. Majority of patients were men (53, 82.8%), came from rural areas (65, 76.4%) and were the main breadwinners (44, 68%) of their family. After the initial trauma, a vast majority of the patients (68, 80%) visited the nearest eye care provider to obtain treatment and a majority of them (47, 69.1%) reached there within 3 h. The remaining patients (17, 20%) came directly to our center, the base hospital (BH). The mean distance travelled to the BH was 113.6 km. The mean total cost incurred was INR 20107.6 ± 10441.3. Approximately 84.8% of the patients reported a monthly income less than this amount. CONCLUSION: The economic impact of receiving primary definitive care following open globe injuries is higher than the average monthly income of more than 2/3rd of the patients. To replicate the success stories like the cataract outreach programs, there is a need to implement an integrated program encompassing workplace safety, legal protection to primary eye care providers and providing economic support for definitive treatment for the vulnerable population.

Notch signaling mediates olfactory multiciliated cell specification.

Epithelial multiciliated cells (MCCs) use motile cilia to direct external fluid flow, the disruption of which is associated with human diseases in a broad array of organs such as those in the respiratory, reproductive, and renal systems. While many of the signaling pathways that regulate MCC formation in these organ systems have been identified, similar characterization of MCC differentiation in the developing olfactory system has been lacking. Here, using live cell tracking, targeted cell ablation, and temporally-specific inhibition of the Notch signaling pathway, we identify the earliest time window of zebrafish olfactory MCC (OMCC) differentiation and demonstrate these cells' derivation from peridermal cells. We also describe regionally segregated Notch signaling across time points of rapid OMCC differentiation and show that Notch signaling downregulation yields an increase in OMCCs, suggesting that OMCC fate is normally repressed in a region-specific manner during olfactory development. Finally, we describe Notch signaling's regulation of the differentiation/ciliogenesis-associated genes foxj1a and foxj1b. Taken together, these findings provide new insights into the origins and developmental programming of OMCCs in vivo.

Neuroblastoma differentiation in vivo excludes cranial tumors.

Neuroblastoma (NB), the most common cancer in the first year of life, presents almost exclusively in the trunk. To understand why an early-onset cancer would have such a specific localization, we xenotransplanted human NB cells into discrete neural crest (NC) streams in zebrafish embryos. Here, we demonstrate that human NB cells remain in an undifferentiated, tumorigenic state when comigrating posteriorly with NC cells but, upon comigration into the head, differentiate into neurons and exhibit decreased survival. Furthermore, we demonstrate that this in vivo differentiation requires retinoic acid and brain-derived neurotrophic factor signaling from the microenvironment, as well as cell-autonomous intersectin-1-dependent phosphoinositide 3-kinase-mediated signaling, likely via Akt kinase activation. Our findings suggest a microenvironment-driven explanation for NB's trunk-biased localization and highlight the potential for induced differentiation to promote NB resolution in vivo.