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Decades of studies have revealed molecular and neural circuit bases for body weight homeostasis. Neural hormone oxytocin (Oxt) has received attention in this context because it is produced by neurons in the paraventricular hypothalamic nucleus (PVH), a known output center of hypothalamic regulation of appetite. Oxt has an anorexigenic effect, as shown in human studies, and can mediate satiety signals in rodents. However, the function of Oxt signaling in the physiological regulation of appetite has remained in question, because whole-body knockout (KO) of Oxt or Oxt receptor (Oxtr) has little effect on food intake. We herein show that acute conditional KO (cKO) of Oxt selectively in the adult PVH, but not in the supraoptic nucleus, markedly increases body weight and food intake, with an elevated level of plasma triglyceride and leptin. Intraperitoneal administration of Oxt rescues the hyperphagic phenotype of the PVH Oxt cKO model. Furthermore, we show that cKO of Oxtr selectively in the posterior hypothalamic regions, especially the arcuate hypothalamic nucleus, a primary center for appetite regulations, phenocopies hyperphagic obesity. Collectively, these data reveal that Oxt signaling in the arcuate nucleus suppresses excessive food intake.

SummaryThe adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naïve male mice are aggressive to the conspecific young, they start to provide caregiving to infants around the time when their own young are expected. How such behavioral plasticity is implemented at the level of neural connections remains poorly understood. Using viral-genetic approaches, here we establish hypothalamic oxytocin neurons as key regulators of parental caregiving behaviors of male mice. We then used rabies virus-mediated unbiased screen to identify excitatory neural connections originating from the lateral hypothalamus to the oxytocin neurons to be drastically strengthened when male mice become fathers. These connections are functionally relevant, as their activation suppresses pup-directed aggression in virgin males. These results demonstrate the life-stage associated, long-distance, and cell-type-specific plasticity of neural connections in the hypothalamus, the brain region classically assumed to be hard-wired.Highlight–OT is indispensable for parental caregiving behavior of male mice–Activation of OT neurons triggers paternal caregiving behavior in otherwise infanticidal sexually-naïve male mice partly via OT ligand–Unbiased rabies virus-mediated screening reveals enhanced connectivity originated from excitatory LHA neurons to OT neurons in fathers.–This structural plasticity can support behavioral plasticity

SummaryPulsatile release of the hormone oxytocin (OT) mediates uterine contraction during parturition and milk ejection during lactation1–3. These pulses are generated by unique activity patterns of the central neuroendocrine OT neurons located in the paraventricular and supraoptic hypothalamus. Classical studies have characterized putative OT neurons by in vivo extracellular recording techniques in rats and rabbits under anesthesia1, 4–7 or awake8–10. Due to technical limitations, however, the identity of OT neurons in these previous studies was speculative based on their electrophysiological characteristics and axonal projection to the posterior pituitary, not on OT gene expression. To pinpoint OT neural activities among other hypothalamic neurons that project to the pituitary11, 12 and make better use of cell-type-specific neuroscience toolkits13, a mouse model needs to be developed for studies of parturition and lactation. We herein introduce viral genetic approaches in mice to characterize the maternal activities of OT neurons by fiber photometry. During lactation, a sharp photometric peak of OT neurons appeared at approximately 520 s following simultaneous suckling stimuli from three pups. The amplitude of the peaks increased as the mother mice experienced lactation, irrespective of the age of the pups, suggesting the intrinsic plasticity of maternal OT neurons. Based on a mono-synaptic input map to OT neurons, we pharmacogenetically activated the inhibitory neurons in the bed nucleus of the stria terminalis and found suppression of the activities of OT neurons. Collectively, our study illuminates temporal dynamics in the maternal neural activities of OT neurons and identifies one of its modulatory circuits.Highlights- Pulsatile activities of genetically-defined OT neurons in mother mice were recorded in vivo.- The maternal experience-dependent plasticity of the OT neural activities was found.- Input-mapping of OT neurons in mother mice was performed by rabies-mediated trans-synaptic tracing.- Photometric peaks of OT neurons were suppressed by the activation of BST inhibitory neurons.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Purpose We aimed to establish purification and culture systems for retinal ganglion cells (RGCs) differentiated from mouse and human pluripotent stem cells (PSC) for in vitro and regenerative medicine studies. Methods We used a two-step immunopanning method to purify RGCs from mouse and human PSC-derived three-dimensional (3D) retinal organoids. To assess the method, we purified RGCs from 3D retinal organoids derived from embryonic stem cells (ESCs) generated from Thy1-EGFP transgenic (TG) mice. In addition, 3D retinal organoids differentiated from human induced PSCs (iPSCs) were cultured for up to differentiation day (DD) 120, and RGCs were purified by immunopanning. RGC marker expressions were confirmed by immunostaining and reverse transcription-quantitative PCR. The purified RGCs were cultured, and neurite outgrowth was measured and analyzed using an IncuCyte Zoom system. Results Mouse RGCs purified from Thy1-EGFP TG mouse retinas and the ESC-derived 3D retinas could be maintained for approximately 2 to 3 weeks, expressing the markers BRN3B and SMI-312. Purified RGCs from human iPSC-derived retinal organoids expressed RGC markers and could be maintained for up to 4 weeks. The RGCs collected at DD 90 to 110 extended longer neurites than those collected at younger stages. Conclusions We successfully purified RGCs from mouse and human PSC-derived 3D retinal organoids cultured for approximately 120 days. RGCs from older retinal organoids would be useful for neurite tracking. This method would be effective not only for studying the pathology of human RGC diseases but also for therapeutic drug studies and RGC transplantation.

Virus inactivation is a critical step in the manufacturing of recombinant therapeutic proteins, in particular antibodies, using mammalian expression systems. We have shown in the previous paper that arginine is effective in inactivation of herpes simplex virus type 1 (HSV-1) and influenza virus at low temperature under mildly acidic pH, i.e., above pH 4.0; above this pH, conformational changes of most antibodies are negligible. We have here extended virus inactivation study of arginine to other enveloped viruses, such as Sendai virus and Newcastle Disease Virus (NDV), and observed that arginine was ineffective against both viruses under the similar conditions, i.e., on ice and above pH 4.0. However, an arginine derivative, butyroyl-arginine, showed a strong virucidal potency against Sendai virus, leading to a 4 log reduction in virus yield at pH 4.0, but not against NDV. In addition, although arginine and butyroyl-arginine were equally effective against influenza virus having a cleaved form of hemagglutinin spike proteins, only butyroyl-arginine was significantly effective against the same virus, but having an uncleaved hemagglutinin spike proteins. Furthermore, butyroyl-arginine was more effective than arginine against HSV-1 at pH 4.5; i.e., it has a broader pH spectrum than does arginine.

Both hot water extracts of coffee grinds and instant coffee solutions inhibited the multiplication of herpes simplex virus type 1, a representative enveloped DNA virus, when they were added to the culture medium of the virus-infected cells at a dose of one fifth the concentration suitable for drinking. The antiherpetic activity was independent of the suppliers (companies) of the coffee grinds and of the locations where the coffee beans were produced. Further characterization revealed that there are two different mechanisms, by which the coffee extracts exert inhibitory activities on the virus infection; (1) a direct inactivation of the infectivity of virus particle (i.e., a virucidal activity) and (2) the inhibition of progeny infectious virus formation at the late stage of viral multiplication in the infected cells. Caffeine, but not quinic acid and chlorogenic acid, inhibited the virus multiplication to some extent, but none of them showed the virucidal activity, suggesting that other component(s) in the coffee extracts must play a role in the observed antiviral activity. In addition, the coffee extracts inhibited the multiplication of poliovirus, a non-enveloped RNA virus, but showed no virucidal effect on this virus.

Arginine, among the amino acids, has demonstrated unique properties, including suppression of protein-protein interactions and virus inactivation. We investigated the effects of arginine on the infectivity of human herpesvirus 2 (HHV-2) and the potential application of arginine as a chemotherapeutic agent against genital herpes. Arginine directly inactivated HHV-2 and characterization of the inactivation demonstrated that 1 M arginine at pH 4.3 inactivated the virus more efficiently compared to 0.1 M citrate or 1 M sodium chloride, indicating that neither acidic pH nor ionic strength alone is sufficient for virus inactivation. The effect of arginine was rapid and concentration-dependent. Although virus inactivation was efficient at an acidic pH, arginine inactivated the virus even at a neutral pH, provided that a higher arginine concentration and prolonged incubation time were used. In addition, arginine suppressed the multiplication of HHV-2 under the conditions at which its effect on cell viability was insignificant. Pilot mouse model studies revealed a marked suppression of death by arginine when the mice were infected with HHV-2 through the vaginal route, followed by an intermittent application of acidic arginine by vaginal instillation.

Various amino acid-derived compounds, for example, Nα-Cocoyl-L-arginine ethyl ester (CAE), alkyloxyhydroxylpropylarginine, arginine cocoate, and cocoyl glycine potassium salt (Amilite), were examined for their virucidal activities against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), influenza A virus (IAV), and poliovirus type 1 (PV-1) in comparison to benzalkonium chloride (BKC) and sodium dodecylsulfate (SDS) as a cationic and anionic control detergent and also to other commercially available disinfectants. While these amino acid-derived compounds were all effective against HSV-1 and HSV-2, CAE and Amilite were the most effective. These two compounds were, however, not as effective against IAV, another enveloped virus, as against HSV. Cytotoxicity of CAE was weak; at 0.012%, only 5% of the cells were killed under the conditions, in which 100% cells were killed by either SDS or BKC. In addition to these direct virucidal effects, CAE inhibited the virus growth in the HSV-1- or PV-1-infected cells even at 0.01%. These results suggest a potential application of CAE as a therapeutic or preventive medicine against HSV superficial infection at body surface.

The present study examined the effect of caffeine on RNA and DNA viruses, revealing that it inhibits the multiplication of both. In the presence of caffeine, the progeny virus yield of both herpes simplex virus type 1 (HSV-1) and poliovirus decreased with increasing concentrations of the reagent, although HSV-1 was much more sensitive than poliovirus. The influenza virus was not affected by caffeine at the same concentrations. None of the viruses were directly inactivated by caffeine at the tested concentrations. Characterization of the mode of action of caffeine against HSV-1 infection revealed that the addition of the reagent at 10 h post-infection significantly affected the formation of progeny virus, indicating that caffeine can inhibit the multiplication of HSV-1 during the step(s) following the completion of viral DNA replication and the formation of nucleocapsids. In addition, the reagent selectively enhanced the cytopathic effects and cell death of the infected cells over uninfected cells, suggesting that the antiviral action of caffeine against HSV-1 is, at least in part, the result of accelerated degeneration of the infected cells.

Hot water extracts of coffee grinds and commercial instant coffee solutions have been shown to exhibit marked antiviral and virucidal activities against herpes simplex virus type 1 (HSV-1). Specifically, it has been shown that caffeine and N-methyl-pyridinium formate inhibit the multiplication of HSV-1 in HEp-2 cells. The present study examined the virological properties and the antiviral activity of caffeic acid against HSV-1. Caffeic acid inhibited the multiplication of HSV-1 in vitro, while chlorogenic acid, a caffeic acid ester with quinic acid, did not. These reagents did not have a direct virucidal effect. The one-step growth curve of HSV-1 showed that the addition of caffeic acid at 8 h post infection (h p.i.) did not significantly affect the formation of progeny viruses. An analysis of the influence of the time of caffeic acid addition, revealed that addition at an early time post infection remarkably inhibited the formation of progeny infectious virus in the infected cells, but its addition after 6 h p.i. (i.e., the time of the completion of viral genome replication) did not efficiently inhibit this process. These results indicate that caffeic acid inhibits HSV-1 multiplication mainly before the completion of viral DNA replication, but not thereafter. Although caffeic acid showed some cytotoxicity by prolonged incubation, the observed antiviral activity is likely not the secondary result of the cytotoxic effect of the reagent, because the inhibition of the virus multiplication was observed before appearance of the notable cytotoxicity.

Acidic pH is frequently used to inactivate viruses. We have previously shown that arginine synergizes with low pH in enhancing virus inactivation. Considering a potential application of the acid inactivation of viruses for the prevention and treatment of superficial virus infection at body surfaces and fixtures, herein we have examined the effects of various electrolytes on the acid-induced inactivation of the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), the influenza A virus (IAV) and the poliovirus upon their incubation at 30˚C for 5 min. Eight electrolytes, i.e., phosphate, NaCl, glutamate, aspartate, pyrrolidone carboxylate, citrate, malate and acetate were tested. No detectable inactivation of the poliovirus was observed under the conditions examined, reflecting its acid-resistance. HSV-1 and HSV-2 responded similarly to the acid-treatment and electrolytes. Some electrolytes showed a stronger virus inactivation than others at a given pH and concentration. The effects of the electrolytes were virus-dependent, as IAV responded differently from HSV-1 and HSV-2 to these electrolytes, indicating that certain combinations of the electrolytes and a low pH can exert a more effective virus inactivation than other combinations and that their effects are virus-specific. These results should be useful in designing acidic solvents for the inactivation of viruses at various surfaces.

IN THE PRESENT STUDY, DEHYDROASCORBIC ACID INHIBITED THE MULTIPLICATION OF VIRUSES OF THREE DIFFERENT FAMILIES: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Although dehydroascorbic acid showed some cytotoxicity at higher concentrations, the observed antiviral activity was not the secondary result of the cytotoxic effect of the reagent, as the inhibition of virus multiplication was observed at reagent concentrations significantly lower than those resulting in cytotoxicity. Characterization of the mode of the antiviral action of dehydroascorbic acid against HSV-1 revealed that the addition of reagent at any time post infection inhibited the formation of progeny infectious virus in the infected cells, and a one-step growth curve showed that the addition of reagent allowed formation for an additional 2 h, but then almost completely suppressed it. These results indicate that the reagent inhibits HSV-1 multiplication after the completion of viral DNA replication, probably at the step of the envelopment of viral nucleocapsids at the Golgi apparatus of infected cells.

N-methyl-pyridinium formate, a novel component of coffee extracts, inhibited the multiplication of both DNA and RNA viruses. In the presence of the compound, the progeny viral yields of both herpes simplex virus type 1 (HSV-1) and poliovirus in HEp-2 cells and those of influenza virus type A in MDCK cells decreased with increasing concentrations of the compound, although the degree of viral sensitivity to this compound differed. In addition, none of these viruses were directly inactivated by the compound at the concentrations tested. Characterization of the mode of action of this compound against HSV-1 multiplication revealed that it inhibits the viral growth primarily at the initial step of virus multiplication, i.e., within 2 h after the onset of multiplication, although the virus multiplication was affected by the compound throughout the multiplication cycle. In addition, this compound showed a significant cytotoxic effect, although the observed antiviral effect was unlikely to be attributed to the cytotoxic effect.

Viral clearance is a primary concern for parenteral protein biopharmaceuticals. Low pH, detergent/solvent wash, or heating, called pasteurization, has been the main process for virus inactivation. Detergent/solvent wash is also used to treat superficial infectious diseases, including herpes simplex virus (HSV) infections. Herein we examined virus inactivation effects of acidic arginine on HSV type 2 (HSV-2) as a function of pH and temperature in an attempt to find solvent conditions that are effective for virus inactivation, yet are compatible with in vivo applications. Aqueous arginine at 0.7 M was highly effective on HSV-2, more so at lower pH and higher temperature. Its effects were stronger than 0.1 M citrate, 0.1 M citrate/0.6 M NaCl or 0.7 M citrate at any pH and temperature. This demonstrates that strong virus inactivation effects of arginine are not simply due to ionic strength or high concentration and arginine possesses a unique property that results in irreversible damage in virus particles. Such strong virus inactivation effects can be used in vivo for certain superficial infectious diseases, such as genital infections.

We investigated the effects of arginine on the multiplication of herpes simplex virus type 1 (HSV-1) and the potential of arginine as an antiherpetic agent. Arginine suppressed the growth of HSV-1 concentration-dependently. Inhibition of HSV-1 by arginine leveled off at 50-60 mM, although the higher concentration was not suitable as an antiviral agent due to cytotoxicity. 'Time of addition' experiments revealed that arginine was particularly effective when added within 6 h post-infection (h p.i.), suggesting that the reagent sensitive step is in the early stages of the infection. A one-step growth curve of HSV-1 in the presence of 30 mM arginine revealed that: i) the latent period was significantly extended, ii) the rate of formation of progeny infectious virus decreased and iii) the final yield of progeny virus decreased to 1%. The addition of arginine at 8 h p.i., after the completion of viral DNA replication in the virus multiplication, allowed the normal formation of progeny virus in the subsequent 4 h, confirming that arginine does not directly interfere with the formation of progeny infectious virus. In addition, arginine also inhibits several RNA viruses.

Elevated temperatures have been used to inactivate viruses for plasma-derived biopharmaceuticals. This paper describes the effects of arginine and NaCl in conjunction with elevated temperature for inactivation of two enveloped viruses, i.e., herpes simplex virus type 1 (HSV-1) and influenza virus type A at neutral pH. In phosphate-buffered saline, a significant inactivation of HSV-1 occurred above 40 degrees C, resulting in less than 10% surviving virus (over 90% virus inactivation) at 50 degrees C. Arginine concentration dependently decreased the temperature required for virus inactivation, leading to temperature shift by almost 17 degrees C at 1.2M. NaCl also decreased the inactivation temperature, but to a considerably lesser extent, indicating that virus inactivation effect of arginine is not simply due to ionic strength. Influenza virus was also inactivated by high temperature, but its responses to arginine and NaCl were different from those on HSV-1, suggesting that virus inactivation mechanism is different between these two viruses, i.e., the effects of these reagents are virus specific.

Virus inactivation is a key step for the purification of pharmaceutical proteins derived from recombinant mammalian expression systems and conventionally done using low pH-treatment, which is often harmful to the proteins to be purified. This is particularly true for antibodies, because immunoglobulin proteins undergo conformational changes at acidic pH. We have been developing mild elution solvents using arginine for Protein-A chromatography to minimize the low pH-induced damages on the antibodies. Here we have tested the aqueous solutions containing arginine or butyroyl-arginine at or above pH 4.0 for their effects on virus inactivation, since these solvents are effective above pH 4.0 in elution of bound antibodies from Protein-A columns. When the virus was incubated on ice, 0.1 M sodium citrate was totally ineffective above pH 4.0, but aqueous solutions containing arginine above 0.35 M or butyroyl-arginine above 0.28 M showed extensive virus killing at or even above pH 4.0.