Mitochondrial inheritance in haploid × non-haploid crosses in Cryptococcus neoformans

In the basidiomycetous yeast Cryptococcus neoformans, fusants and meiotic progeny from haploid–haploid (HH) crosses between strains of mating type a (MAT a) and mating type alpha (MATα) typically inherit mitochondrial DNA (mtDNA) from the MAT a parent. In this study, we investigated the mtDNA inheritance pattern in haploid × non-haploid crosses. A total of 420 meiotic progeny and 173 fusants were obtained from five crosses and analyzed for two polymorphic mitochondrial markers. The percentage of meiotic progeny and fusants inheriting mtDNA from MATα or MATα/α parents ranged from 8 to 50%. The leakage was significantly greater than those observed in HH crosses, indicating that mtDNA inheritance is not uniparental in haploid × non-haploid crosses in C. neoformans. In addition, mtDNA leakage in the fusants, but not the meiotic progeny, of the MATα/α × MAT a cross was significantly higher than that in the MAT a/a × MATα cross, suggesting that the diploid parents with different mating types contribute differently in determining fusant mtDNA genotype in these crosses. Flow cytometry analysis revealed that meiotic progeny population of each cross was of mixed ploidy while the ploidy level of the selected fusants ranged from diploid to triploid.     

Multi-functional graphene as an in vitro and in vivo imaging probe

A strategy has been developed for the synthesis of multi-functional graphene (MFG) using green synthetic approach and explored its biomedical application as a promising fluorescent marker for in vitro and in vivo imaging. In-situ microwave-assisted reduction and magnetization process was adopted to convert the graphene oxide into magnetic graphene within 1 min, which was further covalently modified to build a polyacrylic acid (PAA) bridge for linking the fluorescein o-methacrylate (FMA) to yield MFG with water-dispersibility (∼2.5 g/l) and fluorescence property (emission maximum at 526 nm). The PAA bridges also functions to prevent graphene-induced fluorescence quenching of conjugated FMA. The extent of reduction, magnetization, and functionalization was confirmed with TEM, AFM, Raman, XPS, FT-IR, TGA, and SQUID measurements. In vitro cytotoxicity study of HeLa cells reveal that MFG could stand as a biocompatible imaging probe with an IC₅₀ value of ∼100 μg/ml; whereas in vivo zebrafish study does not induce any significant abnormalities nor affects the survival rate after microinjection of MFG. Confocal laser scanning microscopy images reveals that MFG locates only in the cytoplasm region and exhibits excellent co-localization and biodistribution from the head to tail in the zebrafish. Our results demonstrate the applicability of graphene based fluorescence marker for intracellular imaging and, more significantly, as well as whole-animal imaging. Hence, MFG could preferentially serve as a dual functional probe in biomedical diagnostics.     

RNA interference inhibits yellow fever virus replication in vitro and in vivo

RNA interference (RNAi) is a process that is induced by double stranded RNA and involves the degradation of specific sequences of mRNA in the cytoplasm of the eukaryotic cells. It has been used as an antiviral tool against many viruses, including flaviviruses. The genus Flavivirus contains the most important arboviruses in the world, i.e., dengue (DENV) and yellow fever (YFV). In our study, we investigated the in vitro and in vivo effect of RNAi against YFV. Using stable cell lines that expressed RNAi against YFV, the cell lines were able to inhibit as much as 97% of the viral replication. Two constructions (one against NS1 and the other against E region of YFV genome) were able to protect the adult Balb/c mice against YFV challenge. The histopathologic analysis demonstrated an important protection of the central nervous system by RNAi after 10 days of viral challenge. Our data suggests that RNAi is a potential viable therapeutic weapon against yellow fever.

Indocyanine green-loaded biodegradable tumor targeting nanoprobes for in vitro and in vivo imaging

Indocyanine green (ICG) is a near-infrared (NIR) ?uorescence dye for extensive biological application, but limited by its poor aqueous stability in?vitro, concentration-dependent aggregation, rapid elimination from the body, and lack of target specificity. In this paper, to overcome these limitations, folate receptor-targeted, ICG dye-doped poly(d,l-lactide-co-glycolide) (PLGA) lipid nanoparticles (FA-ICG-PLGA-lipid NPs) were constructed by a single-step self-assemble and nanoprecipitation method. The prepared FA-ICG-PLGA-lipid NPs exhibited good biocompatibility, monodispersity, excellent NIR penetration ability, significant stability against photobleaching and long circulation time. The intracellular uptake experiment proved the targeting efficacy of the FA-ICG-PLGA-lipid NPs was more effective in folate receptor over-expressing MCF-7 cells than folate receptor negative A549 cells. Furthermore, the in?vivo experiments showed the FA-ICG-PLGA-lipid NPs were specifically targeted to the tumor, and its circulation time was much longer than free ICG. These biocompatible and biodegradable NIR-NPs prove a potential application in tumor diagnosis and targeted imaging due to its high aqueous stability, excellent NIR optical properties and significantly targeting property in?vivo.     

Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro

The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen -Folate Receptor (-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.     

Antihistamine therapy in allergic rhinitis

Antihistamines have long been a mainstay in the therapy for allergic rhinitis. Many different oral antihistamines are available for use, and they are classified as first generation or second generation based on their pharmacologic properties and side-effect profiles. The recent introduction of intranasal antihistamines has further expanded the role of antihistamines in the treatment of allergic rhinitis. Certain patient populations, such as children and pregnant or lactating women, require special consideration regarding antihistamine choice and dosing as part of rhinitis therapy.     

Ultrabright and ultrastable near-infrared dye nanoparticles for in vitro and in vivo bioimaging

We report a new strategy of using carrier-free pure near-infrared (NIR) dye nanoparticles (NPs) to achieve highly luminescent NIR fluorescent probes for in?vitro and in?vivo imaging. Bis(4-(N-(2-naphthyl)phenylamino) phenyl)-fumaronitrile (NPAPF) NPs are shown to exhibit favorable biocompatibility, wide-range pH stability (pH 4-10) and much more superior photostability than conventional dyes. Importantly, the combined merits of high dye loading content and aggregation-induced emission enhancement properties, endow the NIR probes with high brightness and a high quantum yield up to 14.9%. The NPAPF NPs can be readily conjugated with folic acid for targeted in?vitro cell imaging. Applications of the NPs probes in high efficiency in?vivo and ex?vivo imaging were successfully demonstrated. Intense fluorescent signals of NPAPF NPs can be distinctly, selectively and spatially resolved in tumor sites with ultrahigh sensitivity, even with 5?ms exposure time, due to the preferentially accumulation of NPs in tumor sites through passive enhanced permeability and retention effect. The totality of results clearly demonstrate the exciting potential of the functionalized NPAPF NPs as a NIR fluorescent probe for in?vitro and in?vivo imaging and diagnostics.     

Effects of fibrin pad hemostat on the wound healing process in vivo and in vitro

Fibrin Pad is a hemostatic pad designed to control surgical-related bleeding. It consists of a fully absorbable composite matrix scaffold coated with human-derived active biologics that immediately form a fibrin clot upon contact with targeted bleeding surfaces. Studies were conducted to investigate the effect of Fibrin Pad and its biologics-free composite matrix component (Matrix) on the wound healing process in in vitro and in vivo models. Fibrin Pad was evaluated in solid organ, soft tissue defects, and subcutaneous tissues. Immunocompromised rodents were used to avoid xeno-mediated responses. Extracts created from both materials were evaluated for biological activity using in vitro cell culture assays. Neither Fibrin Pad nor Matrix alone showed any inhibition of the wound healing of treated defect sites. An apparent accelerated healing was noted in the soft tissue and subcutaneous tissue defects with Fibrin Pad as compared to Matrix. Both materials showed desirable properties associated with tissue scaffolds. The in vitro study results show that Fibrin Pad extract can induce dose-dependent increases in fibroblast proliferation and migration. These studies confirm that the biologic components of Fibrin Pad can enhance wound healing processes in in vitro assays and fully support wound healing at the site of in vivo application.     

DNA methylation episignature in Gabriele-de Vries syndrome

PurposeGabriele-de Vries syndrome (GADEVS) is a rare genetic disorder characterized by developmental delay and/or intellectual disability, hypotonia, feeding difficulties, and distinct facial features. To refine the phenotype and to better understand the molecular basis of the syndrome, we analyzed clinical data and performed genome-wide DNA methylation analysis of a series of individuals carrying a YY1 variant.MethodsClinical data were collected for 13 individuals not yet reported through an international call for collaboration. DNA was collected for 11 of these individuals and 2 previously reported individuals in an attempt to delineate a specific DNA methylation signature in GADEVS.ResultsPhenotype in most individuals overlapped with the previously described features. We described 1 individual with atypical phenotype, heterozygous for a missense variant in a domain usually not involved in individuals with YY1 pathogenic missense variations. We also described a specific peripheral blood DNA methylation profile associated with YY1 variants.ConclusionWe reported a distinct DNA methylation episignature in GADEVS. We expanded the clinical profile of GADEVS to include thin/sparse hair and cryptorchidism. We also highlighted the utility of DNA methylation episignature analysis for classification of variants of unknown clinical significance.     

In vitro and in vivo antibacterial activity of tigecycline against Vibrio vulnificus

Background/purpose

The aim of this study is to investigate the role of tigecycline in Vibrio vulnificus infection.

Enzyme Replacement Therapy in a Gaucher Family

Gaucher disease is a lipid storage disorder due to deficiency of beta glucocerebrosidase. It's an autosomal recessive disease and as a result of this enzyme deficiency, glucocerebroside accumulates in various types of tissues like liver, brain spleen and bone marrow. We aimed to describe the effects of enzyme replacement therapy in three members of a family with Gaucher disease and to emphasize screening of the family members of the patients with Gaucher disease. Furthermore, late diagnosis and treatment in these patients have a minimal effect on improvement of the quality of life, and early diagnosis and treatment are very important in Gaucher disease.     

Dendritic cell and macrophage heterogeneity in vivo

Macrophage and dendritic cell (DC) are hematopoietic cells found in all tissues in the steady state that share the ability to sample the environment but have distinct function in tissue immunity. Controversies remain on the best way to distinguish macrophages from DCs in?vivo. In this Perspective, we discuss how recent discoveries in the origin of the DC and macrophage lineage help establish key functional differences between tissue DC and macrophage subsets. We also emphasize the need to further understand the functional heterogeneity of the tissue DC and macrophage lineages to better comprehend the complex role of these cells in tissue homeostasis and immunity.     

Functional and epigenetic studies reveal multistep differentiation and plasticity of in vitro-generated and in vivo-derived follicular T helper cells

Follicular T helper (Tfh) cells provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated protein (SAP) prevent GC formation because of defective T?cell-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in?vitro differentiation of functionally competent "Tfh-like" cells that expressed interleukin-21, Tfh cell markers, and Bcl6 and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wild-type, yet failed to support GCs in?vivo. Interestingly, both Tfh-like and in?vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2, and Th17 cells could be reprogrammed to obtain Tfh cell characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3, and Rorc in Tfh-like and ex?vivo Tfh cells and on Bcl6 in non-Tfh cells, supporting the concept of plasticity between Tfh and other Th cell populations.     

Cortisol and sleep in infancy and early childhood

Introduction

Cortisol release is often associated with physiological arousal or perceived stress. Findings in adults as well as in older children and adolescents show that cortisol is also connected to sleep. Furthermore, it is assumed that high-quality sleep is a predictor of regular cortisol release throughout the day.

Objective

This review summarizes the current literature on how sleep and cortisol levels are associated in early childhood.

Methods

In order to identify studies on sleep and cortisol in young children, a structured literature search was performed in the PsychINFO, PsycARTICLES, PSYNDEX, and Google Scholar databases.

Results

A total of 14 studies could be included this review. According to the results of the reviewed publications, daily cortisol release patterns develop within the first 6 months of life. In addition, young children display a cortisol awakening response (CAR) and cortisol levels are influenced by taking naps during the day as well as by the quality of nighttime sleep.

Conclusion

After reviewing the recent findings in the literature concerning children from birth up to the age of 5 years, it can be assumed that sleep patterns and sleep are associated with cortisol secretion in early childhood. This finding could be included in the creation and further development of interventional sleep training programs.

     

Drug resistance and genetic mapping in Plasmodium falciparum

Drug resistance in malaria parasites is a serious public health burden, and resistance to most of the antimalarial drugs currently in use has been reported. A better understanding of the molecular mechanisms of drug resistance is urgently needed to slow or circumvent the spread of resistance, to allow local treatments to be deployed more effectively to prolong the life span of the current drugs, and to develop new drugs. Although mutations in genes determining resistance to drugs such as chloroquine and the antifolates have been identified, we still do not have a full understanding of the resistance mechanisms, and genes that contribute to resistance to many other drugs remain to be discovered. Genetic mapping is a powerful tool for the identification of mutations conferring drug resistance in malaria parasites because most drug-resistant phenotypes were selected within the past 60 years. High-throughput methods for genotyping large numbers of single nucleotide polymorphisms (SNPs) and microsatellites (MSs) are now available or are being developed, and genome-wide association studies for malaria traits will soon become a reality. Here we discuss strategies and issues related to mapping genes contributing to drug resistance in the human malaria parasite Plasmodium falciparum.