Asymmetric Distributions of Achromatic Bipolar Cells in the Mouse Retina

In the retina, evolutionary changes can be traced in the topography of photoreceptors. The shape of the visual streak depends on the height of the animal and its habitat, namely, woods, prairies, or mountains. Also, the distribution of distinct wavelength-sensitive cones is unique to each animal. For example, UV and green cones reside in the ventral and dorsal regions in the mouse retina, respectively, whereas in the rat retina these cones are homogeneously distributed. In contrast with the abundant investigation on the distribution of photoreceptors and the third-order neurons, the distribution of bipolar cells has not been well understood. We utilized two enhanced green fluorescent protein (EGFP) mouse lines, Lhx4-EGFP (Lhx4) and 6030405A18Rik-EGFP (Rik), to examine the topographic distributions of bipolar cells in the retina. First, we characterized their GFP-expressing cells using type-specific markers. We found that GFP was expressed by type 2, type 3a, and type 6 bipolar cells in the Rik mice and by type 3b, type 4, and type 5 bipolar cells in the Lhx4 mice. All these types are achromatic. Then, we examined the distributions of bipolar cells in the four cardinal directions and three different eccentricities of the retinal tissue. In the Rik mice, GFP-expressing bipolar cells were more highly observed in the nasal region than those in the temporal retina. The number of GFP cells was not different along with the ventral-dorsal axis. In contrast, in the Lhx4 mice, GFP-expressing cells occurred at a higher density in the ventral region than in the dorsal retina. However, no difference was observed along the nasal-temporal axis. Furthermore, we examined which type of bipolar cells contributed to the asymmetric distributions in the Rik mice. We found that type 3a bipolar cells occurred at a higher density in the temporal region, whereas type 6 bipolar cells were denser in the nasal region. The asymmetricity of these bipolar cells shaped the uneven distribution of the GFP cells in the Rik mice. In conclusion, we found that a subset of achromatic bipolar cells is asymmetrically distributed in the mouse retina, suggesting their unique roles in achromatic visual processing.

AVP-eGFP was significantly upregulated by hypovolemia in the parvocellular division of the paraventricular nucleus in the transgenic rats

Arginine vasopressin (AVP) is produced in the paraventricular (PVN) and supraoptic nuclei (SON). Peripheral AVP, which is secreted from the posterior pituitary, is produced in the magnocellular division of the PVN (mPVN) and SON. In addition, AVP is produced in the parvocellular division of the PVN (pPVN), where corticotrophin releasing factor (CRF) is synthesized. These peptides synergistically modulate the hypothalamic-pituitary-adrenal (HPA) axis. Previous studies have revealed that the HPA axis was activated by the hypovolemia. However, the detailed dynamics of AVP in the pPVN under hypovolemic state has not been elucidated. Here, we evaluated the effects of hypovolemia and hyperosmolality on the hypothalamus, using AVP-enhanced green fluorescent protein (eGFP) transgenic rats. Polyethylene glycol (PEG) or 3% hypertonic saline (HTN) was intraperitoneally administered in order to develop hypovolemia or hyperosmolality. AVP-eGFP intensity was robustly upregulated at 3 and 6 h after intraperitoneal (i.p.) administration of PEG or HTN in the mPVN. While in the pPVN, eGFP intensity was significantly increased at 6 h after i.p. administration of PEG with significant induction of Fos-immunoreactive (-ir) neurons. Consistently, eGFP mRNA, AVP hnRNA, and CRF mRNA in the pPVN and plasma AVP and corticosterone were significantly increased at 6 h after i.p. administration of PEG. The results suggest that AVP and CRF syntheses in the pPVN were activated by hypovolemia, resulting in the activation of the HPA axis.

Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.

Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

A new severity classification of lower limb secondary lymphedema based on lymphatic pathway defects in an indocyanine green fluorescent lymphography study

Most protocols for lymphatic imaging of the lower limb conventionally inject tracer materials only into the interdigital space; however, recent studies indicate that there are four independent lymphatic vessel groups (anteromedial, anterolateral, posteromedial, and posterolateral) in the lower limb. Thus, three additional injection sites are needed for lymphatic imaging of the entire lower limb. We aimed to validate a multiple injection designed protocol and demonstrate its clinical benefits. Overall, 206 lower limbs undergoing indocyanine green fluorescent lymphography with the new injection protocol were registered retrospectively. To assess the influence of predictor variables on the degree of severity, multivariable logistic regression models were used with individual known risk factors. Using a generalized linear model, the area under the curve (AUC) of the conventional clinical model, comprising known severity risk factors, was compared with that of the modified model that included defects in the posterolateral and posteromedial groups. Multivariable logistic regression models showed a significant difference for the posteromedial and posterolateral groups. The AUC of the modified model was significantly improved compared to that of the conventional clinical model. Finding defects in the posteromedial and posterolateral groups is a significant criterion for judging lymphedema severity and introducing a new lymphedema severity classification.

A Recombinant System and Reporter Viruses for Papiine Alphaherpesvirus 2

Primate simplex viruses, including Herpes simplex viruses 1 and 2, form a group of closely related herpesviruses, which establish latent infections in neurons of their respective host species. While neuropathogenic infections in their natural hosts are rare, zoonotic transmission of Macacine alphaherpesvirus 1 (McHV1) from macaques to humans is associated with severe disease. Human infections with baboon-derived Papiine alphaherpesvirus 2 (PaHV2) have not been reported, although PaHV2 and McHV1 share several biological properties, including neuropathogenicity in mice. The reasons for potential differences in PaHV2 and McHV1 pathogenicity are presently not understood, and answering these questions will require mutagenic analysis. Here, we report the development of a recombinant system, which allows rescue of recombinant PaHV2. In addition, we used recombineering to generate viruses carrying reporter genes (Gaussia luciferase or enhanced green fluorescent protein), which replicate with similar efficiency as wild-type PaHV2. We demonstrate that these viruses can be used to analyze susceptibility of cells to infection and inhibition of infection by neutralizing antibodies and antiviral compounds. In summary, we created a recombinant system for PaHV2, which in the future will be invaluable for molecular analyses of neuropathogenicity of PaHV2.

Effect of Lipopolysaccharides on Liver Tumor Metastasis of twist1a/krasV12 Double Transgenic Zebrafish

The poor prognosis of patients diagnosed with hepatocellular carcinoma (HCC) is directly associated with the multi-step process of tumor metastasis. TWIST1, a basic helix-loop-helix (bHLH) transcription factor, is the most important epithelial-mesenchymal transition (EMT) gene involved in embryonic development, tumor progression, and metastasis. However, the role that TWIST1 gene plays in the process of liver tumor metastasis in vivo is still not well understood. Zebrafish can serve as a powerful model for cancer research. Thus, in this study, we crossed twist1a+ and kras+ transgenic zebrafish, which, respectively, express hepatocyte-specific mCherry and enhanced green fluorescent protein (EGFP); they also drive overexpression of their respective transcription factors. This was found to exacerbate the development of metastatic HCC. Fluorescence of mCherry and EGFP-labeled hepatocytes revealed that approximately 37.5% to 45.5% of the twist1a+/kras+ double transgenic zebrafish exhibited spontaneous tumor metastasis from the liver to the abdomen and tail areas, respectively. We also investigated the inflammatory effects of lipopolysaccharides (LPS) on the hepatocyte-specific co-expression of twist1a+ and kras+ in double transgenic zebrafish. Following LPS exposure, co-expression of twist1a+ and kras+ was found to increase tumor metastasis by 57.8%, likely due to crosstalk with the EMT pathway. Our results confirm that twist1a and kras are important mediators in the development of metastatic HCC. Taken together, our in-vivo model demonstrated that co-expression of twist1a+/kras+ in conjunction with exposure to LPS enhanced metastatic HCC offers a useful platform for the study of tumor initiation and metastasis in liver cancer.