Impact of endogenous melatonin on rhythmic behaviors, reproduction, and survival revealed in melatonin-proficient C57BL/6J congenic mice.

The hormone melatonin is synthesized from serotonin by two enzymatic reactions (AANAT and ASMT/HIOMT) in the pineal gland following a circadian rhythm with low levels during the day and high levels at night. The robust nightly peak of melatonin secretion is an output signal of the circadian clock to the whole organism. However, so far the regulatory roles of endogenous melatonin in mammalian biological rhythms and physiology processes are poorly understood. Here, we establish congenic mouse lines (>N10 generations) that are proficient or deficient in melatonin synthesis (AH+/+ or AH-/- mice, respectively) on the C57BL/6J genetic background by crossing melatonin-proficient MSM/Ms with C57BL/6J. AH+/+ mice displayed robust nightly peak of melatonin secretion and had significantly higher levels of pineal and plasma melatonin vs AH-/- mice. Using this mice model, we investigated the role of endogenous melatonin in regulating multiple biological rhythms, physiological processes, and rhythmic behaviors. In the melatonin-proficient (AH+/+) mice, the rate of re-entrainment of wheel-running activity was accelerated following a 6-hour phase advance of dark onset when comparted with AH-/- mice, suggesting a role of endogenous melatonin in facilitating clock adjustment. Further in the AH+/+ mice, there was a significant decrease in body weight, gonadal weight and reproductive performance, and a significant increase in daily torpor (a hypothermic and hypometabolic state lasting only hours during adverse conditions). Endogenous melatonin, however, had no effect in the modulation of the diurnal rhythm of 2-[125 I]-iodomelatonin receptor expression in the SCN, free-running wheel behavior in constant darkness, life span, spontaneous homecage behaviors, and various types of social-emotional behaviors. The findings also shed light on the role of endogenous melatonin in mice domestication and provide new insights into melatonin's action in reducing energy expenditure during a food shortage. In summary, the congenic mice model generated in this study offers a significant advantage toward understanding of the role of endogenous melatonin in regulating melatonin receptor-mediated rhythm behaviors and physiological functions.

Leukotriene B4 Receptor Type 2 Accelerates the Healing of Intestinal Lesions by Promoting Epithelial Cell Proliferation.

Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.

Natural Single-Nucleotide Variations in the HIV-1 Genomic SA1prox Region Can Alter Viral Replication Ability by Regulating Vif Expression Levels.

UNLABELLED: We previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally. IMPORTANCE: While human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving forces behind mutual evolution. The interplay of cellular APOBEC3G and viral Vif proteins is a typical example. Here, we demonstrate that naturally occurring single-nucleotide variations in the proximal region of splicing acceptor 1 (SA1prox) of the HIV-1 genome frequently alter Vif expression levels, thereby modulating viral replication potential in cells with various ABOBEC3G levels. The results of the present study reveal a previously unidentified and important way for HIV-1 to compete with APOBEC3G restriction by regulating its Vif expression levels. We propose that SA1prox plays a regulatory role in Vif counteraction against APOBEC3G in order to contribute to HIV-1 replication and evolution, and this may be applicable to other primate lentiviruses.

Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling.

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.

Experimental evidence for core-Merge in the vocal communication system of a wild passerine.

One of the cognitive capacities underlying language is core-Merge, which allows senders to combine two words into a sequence and receivers to recognize it as a single unit. Recent field studies suggest intriguing parallels in non-human animals, e.g., Japanese tits (Parus minor) combine two meaning-bearing calls into a sequence when prompting antipredator displays in other individuals. However, whether such examples represent core-Merge remains unclear; receivers may perceive a two-call sequence as two individual calls that are arbitrarily produced in close time proximity, not as a single unit. If an animal species has evolved core-Merge, its receivers should treat a two-call sequence produced by a single individual differently from the same two calls produced by two individuals with the same timing. Here, we show that Japanese tit receivers exhibit antipredator displays when perceiving two-call sequences broadcast from a single source, but not from two sources, providing evidence for core-Merge in animals.

Delivery of aPD-L1 antibody to i.p. tumors via direct penetration by i.p. route: Beyond EPR effect.

Chemotherapy for peritoneal dissemination is poorly effective owing to limited drug transfer from the blood to the intraperitoneal (i.p.) compartment after intravenous (i.v.) administration. i.p. chemotherapy has been investigated to improve drug delivery to tumors; however, the efficacy continues to be debated. As anticancer drugs have low molecular weight and are rapidly excreted through the peritoneal blood vessels, maintaining the i.p. concentration as high as expected is a challenge. In this study, we examined whether i.p. administration is an efficient route of administration of high-molecular-weight immune checkpoint inhibitors (ICIs) for the treatment of peritoneal dissemination using a model of peritoneal disseminated carcinoma. After i.p. administration, the amount of anti-PD-L1 antibody transferred into i.p. tumors increased by approximately eight folds compared to that after i.v. administration. Intratumoral distribution analysis revealed that anti-PD-L1 antibodies were delivered directly from the i.p. space to the surface of tumor tissue, and that they deeply penetrated the tumor tissues after i.p. administration; in contrast, after i.v. administration, anti-PD-L1 antibodies were only distributed around blood vessels in tumor tissues via the enhanced permeability and retention (EPR) effect. Owing to the enhanced delivery, the therapeutic efficacy of anti-PD-L1 antibody in the peritoneal dissemination models was also improved after i.p. administration compared to that after i.v. administration. This is the first study to clearly demonstrate an EPR-independent delivery of ICIs to i.p. tumors by which ICIs were delivered in a massive amount to the tumor tissue via direct penetration after i.p. administration.

Cellular Reprogramming and Immortality: Expression Profiling Reveals Putative Genes Involved in Turritopsis dohrnii's Life Cycle Reversal.

To gather insight on the genetic network of cell reprogramming and reverse development in a nonmodel cnidarian system, we produced and annotated a transcriptome of the hydrozoan Turritopsis dohrnii, whose medusae respond to damage or senescence by metamorphosing into a juvenile stage (the polyp), briefly passing through an intermediate and uncharacterized stage (the cyst), where cellular transdifferentiation occurs. We conducted sequential and pairwise differential gene expression (DGE) analyses of the major life cycle stages involved in the ontogenetic reversal of T. dohrnii. Our DGE analyses of sequential stages of T. dohrnii's life cycle stages show that novel and characterized genes associated with aging/lifespan, regulation of transposable elements, DNA repair, and damage response, and Ubiquitin-related processes, among others, were enriched in the cyst stage. Our pairwise DGE analyses show that, when compared with the colonial polyp, the medusa is enriched with genes involved in membrane transport, the nervous system, components of the mesoglea, and muscle contraction, whereas genes involved in chitin metabolism and the formation of the primary germ layers are suppressed. The colonial polyp and reversed polyp (from cyst) show significant differences in gene expression. The reversed polyp is enriched with genes involved in processes such as chromatin remodeling and organization, matrix metalloproteinases, and embryonic development whereas suppressing genes involved in RAC G-protein signaling pathways. In summary, we identify genetic networks potentially involved in the reverse development of T. dohrnii and produce a transcriptome profile of all its life cycle stages, and paving the way for its use as a system for research on cell reprogramming.

Transcriptome Characterization of Reverse Development in Turritopsis dohrnii (Hydrozoa, Cnidaria).

Medusae of Turritopsis dohrnii undergo reverse development in response to physical damage, adverse environmental conditions, or aging. Senescent, weakened or damaged medusae transform into a cluster of poorly differentiated cells (known as the cyst stage), which metamorphose back into a preceding life cycle stage, the polyp. During the metamorphosis, cell transdifferentiation occurs. The cyst represents the intermediate stage between a reverting medusa and a healthy polyp, during which cell transdifferentiation and tissue reorganization take place. Here we characterize and compare the transcriptomes of the polyp and newborn medusa stages of T. dohrnii with that of the cyst, to identify biological networks potentially involved in the reverse development and transdifferentiation processes. The polyp, medusa and cyst of T. dohrnii were sequenced through Illumina RNA-sequencing and assembled using a de novo approach, resulting in 92,569, 74,639 and 86,373 contigs, respectively. The transcriptomes were annotated and comparative analyses among the stages identified biological networks that were significantly over-and under-expressed in the cyst as compared to the polyp and medusa stages. Biological processes that occur at the cyst stage such as telomerase activity, regulation of transposable elements and DNA repair systems, and suppression of cell signaling pathways, mitotic cell division and cellular differentiation and development may be involved in T. dohrnii's reverse development and transdifferentiation. Our results are the first attempt to understand T. dohrnii's life-cycle reversal at the genetic level, and indicate possible avenues of future research on developmental strategies, cell transdifferentiation, and aging using T. dohrnii as a non-traditional in vivo system.

Mice modulate ultrasonic calling bouts according to sociosexual context.

Mice produce various sounds within the ultrasonic range in social contexts. Although these sounds are often used as an index of sociability in biomedical research, their biological significance remains poorly understood. We previously showed that mice repeatedly produced calls in a sequence (i.e. calling bout), which can vary in their structure, such as Simple, Complex or Harmonics. In this study, we investigated the use of the three types of calling bouts in different sociosexual interactions, including both same- and opposite-sex contexts. In same-sex contexts, males typically produced a Simple calling bout, whereas females mostly produced a Complex one. By contrast, in the opposite-sex context, they produced all the three types of calling bouts, but the use of each calling type varied according to the progress and mode of sociosexual interaction (e.g. Harmonic calling bout was specifically produced during reproductive behaviour). These results indicate that mice change the structure of calling bout according to sociosexual contexts, suggesting the presence of multiple functional signals in their ultrasonic communication.

The enhancement effect of estradiol on contextual fear conditioning in female mice.

Several studies have reported regulatory effects of estrogens on fear conditioning in female rodents. However, these studies used different doses, durations, and/or administration methods, and reported inconsistent results. To clarify the effect of estrogen on fear conditioning, we investigated the effects of different doses and durations of estradiol administration on freezing behavior during contextual fear conditioning in ovariectomized (OVX) mice. In Experiment 1, OVX ICR mice received a single subcutaneous (s.c.) injection of either oil vehicle (control, 0.1 ml sesame oil) or varied doses (0.5 µg/0.1 ml, 5 µg/0.1 ml, or 50 µg/0.1 ml) of 17ß-estradiol-3-benzoate (EB). Fear conditioning was conducted two days post-EB treatment, and the mice were tested for the learned fear response the following day. In Experiment 2, OVX female mice received an s.c. implantation of a Silastic capsule (I.D. 1.98 × 20.0 mm) containing either vehicle or varied doses (0.05 µg/0.1 ml, 0.5 µg/0.1 ml, 5 µg/0.1 ml, 50 µg/0.1 ml) of EB. Two weeks after implantation, fear conditioning was conducted. During the tests conducted 24 h after conditioning, the high dose EB group showed longer freezing times in both experiments, and lower locomotor activity compared to the control or lower dose groups. In Experiment 3, serum estradiol concentrations of the mice that were treated like those in Experiment 2, were measured; the serum levels of estradiol increased linearly according to the dose of EB administered. The results suggest that mice treated with a high dose of EB exhibit enhanced fear learning, regardless of treatment duration. As a woman's vulnerability to emotional disorders increases in the peripregnancy period, during which estrogen levels are high, the results from the high-dose EB groups may be important for understanding the hormonal mechanisms involved in these disorders.

[Microbial Growth in Unfinished Beverages in Plastic Bottles and the Awareness of Nursing Students in a University about Microbial Contamination].

OBJECTIVES: The purposes of this study were to clarify the microbial growth when drinking a beverage directly from its plastic bottle using models under consumption conditions characteristic for nursing students in a university and their awareness about microbial contamination in unfinished beverages in bottles. METHODS: Three types of bottled beverages were tested: mineral water, Japanese green tea with catechin, and a lactic acid beverage. The ways of drinking were putting the entire lip of a bottle into the mouth (type A) and holding half of the lip out of the mouth (type B). The bottles were kept at room temperature for 8 hours. Samples from unfinished beverages were cultured to detect viable bacteria. An anonymous questionnaire was sent to 324 nursing students of a university, among which 279 responded. RESULTS: The number of viable bacterial cells was larger in type A of drinking than in type B. It increased in mineral water until 2 hours, after which it remained the same. It decreased in Japanese green tea with catechin and the lactic acid beverage. However, even the smallest number exceeded the standard number for drinking beverages. Among the nursing students, 62.7% were aware of microbial contamination in unfinished beverages, but despite this awareness, they did not consume their beverages as soon as possible. CONCLUSION: It is necessary to provide the nursing students with health education on the safety of partially consumed bottled beverages with respect to food hygiene, even if microorganisms do not grow in some types of beverages.

Induction of prolonged natural lifespans in mice exposed to acoustic environmental enrichment.

We investigated the effect of acoustic environmental enrichment (EE) on the lifespans and behaviours of mice to the end of their natural lifespan in different acoustic environments. Acoustic EE induced a significantly prolonged natural lifespan (nearly 17% longer) and was associated with increased voluntary movements. However, no correlation between lifespan and voluntary movements was detected, suggesting that increased voluntary movements are not a primary cause of lifespan prolongation. Analyses of individual differences in lifespan demonstrated that lifespan extension induced by acoustic EE could be related to changes in social relationships (e.g., reduction of social conflict) among individuals kept within a cage. Therefore, an acoustic component may be an important factor inducing the positive effects of EE.

Phase-Specific Vocalizations of Male Mice at the Initial Encounter during the Courtship Sequence.

Mice produce ultrasonic vocalizations featuring a variety of syllables. Vocalizations are observed during social interactions. In particular, males produce numerous syllables during courtship. Previous studies have shown that vocalizations change according to sexual behavior, suggesting that males vary their vocalizations depending on the phase of the courtship sequence. To examine this process, we recorded large sets of mouse vocalizations during male-female interactions and acoustically categorized these sounds into 12 vocal types. We found that males emitted predominantly short syllables during the first minute of interaction, more long syllables in the later phases, and mainly harmonic sounds during mounting. These context- and time-dependent changes in vocalization indicate that vocal communication during courtship in mice consists of at least three stages and imply that each vocalization type has a specific role in a phase of the courtship sequence. Our findings suggest that recording for a sufficiently long time and taking the phase of courtship into consideration could provide more insights into the role of vocalization in mouse courtship behavior in future study.

Effects of amygdala lesions on male mouse ultrasonic vocalizations and copulatory behaviour.

Mice produce ultrasonic vocalizations (USVs) in several behavioural contexts. In particular, male mice articulate a long series of various sounds to females during courtship behaviour. To determine the relationships between this kind of vocal behaviour and emotion, we examined the lesion effects of the amygdala, an important neural module in emotional behaviour, on USVs. We recorded USVs from mice in the lesion and the control (sham operation) groups upon presentation of females and compared USVs before and after surgery. We found that the mean syllable duration of the USVs shortened and the appearance rate of longer syllables decreased after the surgery. The main reasons for these alterations could be explained by the altered courtship behaviour. As reported previously, the mounting behaviour of the lesion group after surgery was markedly less than that of the control group. Therefore, the appearance rate of those longer syllables would decrease logically because longer syllables primarily appear during mounting and intromission. However, we can hypothesize another scenario for the alterations to vocal behaviour: effects on the direct amygdala-periaqueductal grey (PAG) projection might be involved in the increase in the appearance rate of shorter syllables owing to lesion-induced loss of emotions, such as vigilance. Overall, the results suggested two possible mechanisms of the amygdala lesions on the alteration of the vocal behaviour.

Species tropism of HIV-1 modulated by viral accessory proteins.

Human immunodeficiency virus type 1 (HIV-1) is tropic and pathogenic only for humans, and does not replicate in macaque monkeys routinely used for experimental infections. This specially narrow host range (species tropism) has impeded much the progress of HIV-1/acquired immunodeficiency syndrome (AIDS) basic research. Extensive studies on the underlying mechanism have revealed that Vif, one of viral accessory proteins, is critical for the HIV-1 species tropism in addition to Gag-capsid protein. Another auxiliary protein Vpu also has been demonstrated to affect this HIV-1 property. In this review, we focus on functional interactions of these HIV-1 proteins and species specific-restriction factors. In addition, we describe an evolutional viewpoint that is relevant to the species tropism of HIV-1 controlled by the accessory proteins.

Determination of urinary glyoxal and methylglyoxal by high-performance liquid chromatography.

Carbonyl stress compounds such as glyoxal and methylglyoxal have been recently attracting much attention because of their possible clinical significance in chronic and age-related diseases. A high-performance liquid chromatographic procedure has been developed for the simultaneous quantitation of glyoxal and methylglyoxal in human urine. The assay is based on the reaction of these compounds with 1,2-diamino-4,5-dimethoxybenzene to form fluorescent adducts, which are separated by reversed-phase high-performance liquid chromatography in a total run time of 45 minutes and quantitated fluorometrically using 2,3-pentanedione as an internal standard. Derivatization is performed for diluted urine (100-120 mOsm/kg H2O) under acidic conditions (pH 4.5) at 60 degrees C over a prolonged time (15 h) to maximize the yields. The assay is specific and sensitive enough to analyze urinary levels of glyoxal and methylglyoxal with the within- and between-day relative standard deviations of less than 5%. Urinary levels (mean +/- standard deviation, n = 16) of glyoxal and methylglyoxal in healthy subjects were 4.7 +/- 1.35 microg/mg creatinine, 2.2 +/- 0.65 microg/mg creatinine, respectively, the former being 2 to 3 times more than the latter in every subject. The glyoxal and methylglyoxal levels positively correlated with each other, which may suggest that the levels reflect the individual activity of glyoxalase by which both compounds are detoxified.