Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

BACKGROUND: The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. METHODS: nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. RESULTS: In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. CONCLUSIONS: Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

GnRH replacement rescues cognition in Down syndrome.

At the present time, no viable treatment exists for cognitive and olfactory deficits in Down syndrome (DS). We show in a DS model (Ts65Dn mice) that these progressive nonreproductive neurological symptoms closely parallel a postpubertal decrease in hypothalamic as well as extrahypothalamic expression of a master molecule that controls reproduction-gonadotropin-releasing hormone (GnRH)-and appear related to an imbalance in a microRNA-gene network known to regulate GnRH neuron maturation together with altered hippocampal synaptic transmission. Epigenetic, cellular, chemogenetic, and pharmacological interventions that restore physiological GnRH levels abolish olfactory and cognitive defects in Ts65Dn mice, whereas pulsatile GnRH therapy improves cognition and brain connectivity in adult DS patients. GnRH thus plays a crucial role in olfaction and cognition, and pulsatile GnRH therapy holds promise to improve cognitive deficits in DS.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

A dynamic mechanical method to assess bulk viscoelastic behavior of the dentin extracellular matrix.

OBJECTIVES: To develop a protocol for assessment of the bulk viscoelastic behavior of dentin extracellular matrix (ECM), and to assess relationships between induced collagen cross-linking and viscoelasticity of the dentin ECM. METHODS: Dentin ECM was treated with agents to induce exogenous collagen cross-linking: proanthocyanidins (PACs) from Vitis vinifera - VVe, PACs from Pinus massoniana - PMe, glutaraldehyde - (GA), or kept untreated (control). A dynamic mechanical strain sweep method was carried out in a 3-point bending submersion clamp at treatment; after protein destabilization with 4 M urea and after 7-day, 6-month, and 12-month incubation in simulated body fluid. Tan δ, storage (E'), loss (E"), and complex moduli (E*) were calculated and data were statistically analyzed using two-way ANOVA and post-hoc tests (α = 0.05). Chemical analysis of dentin ECM before and after protein destabilization was assessed with ATR-FTIR spectroscopy. RESULTS: Significant interactions between study factors (treatment vs. time points, p < 0.001) were found for all viscoelastic parameters. Despite a significant decrease in all moduli after destabilization, PAC-treated dentin remained statistically higher than control (p < 0.001), indicating permanent mechanical enhancement after biomodification. Covalently crosslinked, GA-treated dentin was unaffected by destabilization (p = 0.873) and showed the lowest damping capacity (tan δ) at all time points (p < 0.001). After 12 months, the damping capacity of PMe and VVe groups decreased significantly. Changes in all amide IR resonances revealed a partial chemical reversal of PAC-mediated biomodification. SIGNIFICANCE: Viscoelastic measurements and IR spectroscopy aid in elucidating the role of inter-molecular collagen cross-linking in the mechanical behavior of dentin ECM.

Targeting Trimeric and Tetrameric Proanthocyanidins of Cinnamomum verum Bark as Bioactives for Dental Therapies.

The present study elucidated the structures of three A-type tri- and tetrameric proanthocyanidins (PACs) isolated from Cinnamomum verum bark to the level of absolute configuration and determined their dental bioactivity using two therapeutically relevant bioassays. After selecting a PAC oligomer fraction via a biologically diverse bioassay-guided process, in tandem with centrifugal partition chromatography, phytochemical studies led to the isolation of PAC oligomers that represent the main bioactive principles of C. verum: two A-type tetrameric PACs, epicatechin-(2ß→O→7,4ß→8)-epicatechin-(4ß→6)-epicatechin-(2ß→O→7,4ß→8)-catechin (1) and parameritannin A1 (2), together with a trimer, cinnamtannin B1 (3). Structure determination of the underivatized proanthocyanidins utilized a combination of HRESIMS, ECD, 1D/2D NMR, and 1H iterative full spin analysis data and led to NMR-based evidence for the deduction of absolute configuration in constituent catechin and epicatechin monomeric units.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

A galloylated dimeric proanthocyanidin from grape seed exhibits dentin biomodification potential.

Grape seeds are a rich source of polyphenols, especially proanthocyanidins (PACs), and are also known for the presence of galloylated oligomeric PACs (OPACs). The present study focuses on the phytochemical methodology for grape seed (O)PACs and their potential role as dentin biomodifiers to be used in restorative and reparative dentistry. A new method using centrifugal partition chromatography (CPC) was developed for the preparative separation of the grape seed (O)PACs. Orthogonal phytochemical profiling of the resulting CPC fractions was performed using C18 and diol HPLC, normal phase HPTLC, and IT-TOF MS analysis. A galloylated procyanidin dimer (1) was isolated from a CPC fraction in order to evaluate its potential to enhance dentin bio-mechanical properties. Moreover, it helped to evaluate the impact of the galloyl moiety on the observed bioactivity. Structure elucidation was performed using ESI-MS, 1D and 2D NMR analyses. For the first time, (1)H iterative full spin analysis (HiFSA) was performed on this type of molecule, enabling a detailed proton chemical shift and coupling constant assignment. The CPC fractions as well as 1 showed promising results in the dentin stiffness bioassay and indicate that they may be used as dental intervention biomaterial.

Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain.

Leptin secreted by adipocytes acts on the brain to reduce food intake by regulating neuronal activity in the mediobasal hypothalamus (MBH). Obesity is associated with resistance to high circulating leptin levels. Here, we demonstrate that peripherally administered leptin activates its receptor (LepR) in median eminence tanycytes followed by MBH neurons, a process requiring tanycytic ERK signaling and the passage of leptin through the cerebrospinal fluid. In mice lacking the signal-transducing LepRb isoform or with diet-induced obesity, leptin taken up by tanycytes accumulates in the median eminence and fails to reach the MBH. Triggering ERK signaling in tanycytes with EGF reestablishes leptin transport, elicits MBH neuron activation and energy expenditure in obese animals, and accelerates the restoration of leptin sensitivity upon the return to a normal-fat diet. ERK-dependent leptin transport by tanycytes could thus play a critical role in the pathophysiology of leptin resistance, and holds therapeutic potential for treating obesity.

Antibacterial potential of citral derivatives.

Nine conjugated acid derivatives from monoterpene citral have been synthesized using ester, amide condensations, Wittig, Knoevenagel, Baylis-Hillman reactions and their antimicrobial activities were evaluated through disc diffusion and serial dilution methods. Several derivatives displayed interesting antimicrobial activity. Particularly, the Baylis-Hillman adducts 8-10 were found to have more potent antibacterial and antifungal activities than standard drugs namely chloramphenicol and nystatin respectively.