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Weedy rice ( Oryza sativa f. spontanea ) presumably originated from rice but has weedy characteristics. It is one of the malignant weeds in rice field that decreases rice production and grain quality severely. In this study, the biological characteristics of weedy rice were described from morphology, shattering, dormancy and stress tolerance. The process of weedy rice origination from de-domestication of cultivated rice was summarized briefly. We also introduced the worldwide distribution of weedy rice as well as its non-uniform distribution in China with the most severe occurrence in middle-south area of Jiangsu province and Zhanjiang of Guangzhou province. In addition, we analyzed the agricultural damage on rice yield, rice quality and paddy field ecology environment by weedy rice. To control the occurrence and spread of weedy rice, we further proposed comprehensive measures, including prevention and controlling measures, reasonable cultivation system, and scientific use of chemical herbicides.

Getting around the blood–brain barrier The meninges comprise three membranes that surround and protect the central nervous system (CNS). Recent studies have noted the existence of myeloid cells resident there, but little is known about their ontogeny and function, and whether other meningeal immune cell populations have important roles remains unclear (see the Perspective by Nguyen and Kubes). Cugurra et al. found in mice that a large proportion of continuously replenished myeloid cells in the dura mater are not blood derived, but rather transit from cranial bone marrow through specialized channels. In models of CNS injury and neuroinflammation, the authors demonstrated that these myeloid cells have an immunoregulatory phenotype compared with their more inflammatory blood-derived counterparts. Similarly, Brioschi et al. show that the meninges host B cells that are also derived from skull bone marrow, mature locally, and likely acquire a tolerogenic phenotype. They further found that the brains of aging mice are infiltrated by a second population of age-associated B cells, which come from the periphery and may differentiate into autoantibody-secreting plasma cells after encountering CNS antigens. Together, these two studies may inform future treatment of neurological diseases. Science , abf7844, abf9277, this issue p. eabf7844 , p. eabf9277 ; see also abj8183, p. 396

Emerging evidence suggests that chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease with disparate inflammatory characteristics between different racial groups and geographies. Currently, little is known about possible underlying distinguishing factors between these inflammatory differences.Our aim was to interrogate differences in CRSwNP disease between White/non-Asian patients and Japanese patients by using whole transcriptome and single-cell RNA gene expression profiling of nasal polyps (NPs).We performed whole transcriptome RNA sequencing with endotype stratification of NPs from 8 White patients (residing in the United States) and 9 Japanese patients (residing in Japan). Reproducibility was confirmed by quantitative PCR in an independent validation set of 46 White and 31 Japanese patients. Single-cell RNA sequencing (scRNAseq) was used to stratify key cell types for contributory transcriptional signatures.Unsupervised clustering analysis identified 2 major endotypes that were present within both cohorts of patients with NPs and had previously been reported at the cytokine level: (1) type 2 endotype and (2) non-type 2 endotype. Importantly, there was a statistically significant difference in the proportion of these endotypes between these geographically distinct subgroups with NPs (P = .03). Droplet-based single-cell RNA sequencing further identified prominent type 2 inflammatory transcript expression: C-C motif chemokine ligand 13 (CCL13) and CCL18 in M2 macrophages, as well as cystatin SN (CST1) and CCL26 in basal, suprabasal, and secretory epithelial cells.NPs from both racial groups harbor the same 2 major endotypes, which we have determined to be present in differing ratios between each cohort with CRSwNP disease. Distinct inflammatory and epithelial cells contribute to the type 2 inflammatory profiles observed.

Two Aplysia command neurons drive motor programs with various levels of variability through synaptic noise and different strength., Behavioral variability often arises from variable activity in the behavior-generating neural network. The synaptic mechanisms underlying this variability are poorly understood. We show that synaptic noise, in conjunction with weak feedforward excitation, generates variable motor output in the Aplysia feeding system. A command-like neuron (CBI-10) triggers rhythmic motor programs more variable than programs triggered by CBI-2. CBI-10 weakly excites a pivotal pattern-generating interneuron (B34) strongly activated by CBI-2. The activation properties of B34 substantially account for the degree of program variability. CBI-10– and CBI-2–induced EPSPs in B34 vary in amplitude across trials, suggesting that there is synaptic noise. Computational studies show that synaptic noise is required for program variability. Further, at network state transition points when synaptic conductance is low, maximum program variability is promoted by moderate noise levels. Thus, synaptic strength and noise act together in a nonlinear manner to determine the degree of variability within a feedforward network.

Background: The future in flexor tendon surgery involves tissue engineering approaches directed toward increasing early repair strength to accelerate tendon healing and to allow for earlier onset of rehabilitation. Previous work has shown that pluripotential mesenchymal stem cells may be successfully delivered to a tendon repair site using a suture carrier. The current work describes the use of Myostatin (GDF-8) to help guide these delivered pluripotential stem cells to differentiate down a tenocyte lineage to potentially maximize the reparative effects of these cells at the tendon repair site.Methods: Primary rat bone marrow mesenchymal stem cells isolated from the long bones of male Sprague-Dawley rats were treated with 500 ng/ml myostatin for 24 h, 48 h, and 72 h. Collagen 1 A, scleraxis (Scx), and tenomodulin (Tnmd) expression, indicative of tenogenesis, was analyzed using real time PCR and immunohistochemistry staining. A migration assay was performed to assess the functional activity of BMSCs after they were treated with myostatin.Results: Compared to the control cells (without treatment), the cells treated with 500 ng/ml myostatin for 72 h exhibited higher expression of Col 1A, Scx, and Tnmd. The mRNA expression of Col1A, Scx, Tnmd increased 15.3, 13 and 7 times respectively. Immunohistochemistry staining showed Scx and Tnmd were expressed in the cellular cytoplasm. In response to myostatin, the cells also showed a tendency to proliferate and migrate more than the control cells.Conclusions: Myostatin (GDF-8) has the ability to increase rat bone marrow mesenchymal stem cell growth and differentiation toward a tenocyte lineage. This information could be useful for future studies regarding tendon repair.

Clear cell renal cell carcinoma (ccRCC) has been associated with one of the highest mortality rates among all cancers. Fatty acid binding proteins (FABPs) are 14‑15 kDa proteins that are highly abundant in the cytosol of most tissues. FABP5, a member of the FABP family, has been observed to promote tumor cell growth in numerous cancer types. In order to investigate the function of FABP5 in ccRCC cells in the present study, RNA sequencing data from The Cancer Genome Atlas were analyzed to determine the expression levels of FABP5 in ccRCC patient samples. Survival and Cox regression analyses were performed to measure the association between FABP5 expression and clinicopathological features of patients with ccRCC. Subsequent in vitro experiments downregulated or overexpressed FABP5 in Caki‑1 and 786O ccRCC cells using lentiviral vectors to evaluate cell proliferation ability, and a xenograft transplantation model was established to examine the effect of FABP5 on tumorigenesis in vivo. The results demonstrated that FABP5 expression was significantly upregulated in samples from patients with ccRCC when compared with normal tissue samples. High FABP5 expression was also significantly correlated with tumor and metastasis classifications and predicted poor survival in patients with ccRCC. In ccRCC cells, silencing of FABP5 significantly inhibited cell proliferation, while overexpression of FABP5 promoted cell proliferation when compared to the respective controls. In addition, treatment with the phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K)/AKT inhibitor, LY294002, attenuated the pro‑proliferative effects of exogenous FABP5 expression in Caki‑1 and 786O cells. This indicated that the PI3K/AKT signaling pathway may be partially involved in the FABP5‑mediated increase in ccRCC cell proliferation. Furthermore, FABP5 was observed to regulate tumor growth in nude mice in vivo. In conclusion, the results of the present study suggest that FABP5 may exert a pro‑proliferative role in ccRCC and may be associated with malignant progression and tumorigenesis.

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.

Sogatella furcifera (Horvath) is a major threat to rice production in China. Insecticides are a key component of typical pest management schemes, but few studies have quantitatively identified insecticide control failures due to insecticide resistance. Therefore, we measured the susceptibility of eight populations to thirteen insecticides by the rice dipping method in 2018 and further evaluated the likelihood of control failure in this study. The results showed that S. furcifera had high levels of S. furcifera resistance to buprofezin (RR = 76.42–160.14-fold), chlorpyrifos (RR = 93.50–219.90-fold), and pymetrozine (RR > 469.30-fold) and low to moderate levels of resistance to nitenpyram (RR = 1.50–10.21-fold), clothianidin (RR = 3.72–20.43-fold), thiamethoxam (RR = 2.88–19.95-fold), imidacloprid (RR = 4.05–31.81-fold), dinotefuran (RR = 4.55–25.98-fold), isoprocarb (RR = 2.55–15.71-fold), and etofenprox (RR = 5.63–17.74-fold). S. furcifera remained susceptible to cycloxaprid (resistance ratio, RR = 0.56–1.82-fold), triflumezopyrim (RR = 0.77–3.23-fold) and sulfoxaflor (RR = 2.28–4.32-fold). Pymetrozine, isoprocarb and etofenprox were determined to be at risk of control failures if the minimum recommended concentration is applied to manage this pest. Furthermore, significant correlations were found among the logarithms of the median lethal concentration (LC50) values of thirteen insecticides. Additionally, the activities of esterases in field populations of S. furcifera were significantly positively correlated with the logarithm of the LC50 values of triflumezopyrim, nitenpyram and clothianidin. The results of this survey offer an important basis for producing effective and useful strategies to manage insecticide resistance in S. furcifera.

l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid-containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid-containing peptide (d2-ATRP) and identified a specific G protein-coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.

Background: We have reported that bioactive sutures coated with bone marrow–derived mesenchymal stem cells (BMSCs) enhance tendon repair strength in an in vivo rat model. We have additionally shown that growth differentiation factor 8 (GDF-8, also known as myostatin) simulates tenogenesis in BMSCs in vitro. The purpose of this study was to determine the possibility of BMSC-coated bioactive sutures treated with GDF-8 to increase tendon repair strength in an in vivo rabbit tendon repair model. Methods: Rabbit BMSCs were grown and seeded on to 4-0 Ethibond sutures and treated with GDF-8. New Zealand white rabbits’ bilateral Achilles tendons were transected and randomized to experimental (BMSC-coated bioactive sutures treated with GDF-8) or plain suture repaired control groups. Tendons were harvested at 4 and 7 days after the surgery and subjected to tensile mechanical testing and quantitative polymerase chain reaction. Results: There were distinguishing differences of collagen and matrix metalloproteinase RNA level between the control and experimental groups in the early repair periods (day 4 and day 7). However, there were no significant differences between the experimental and control groups in force to 1-mm or 2-mm gap formation or stiffness at 4 or 7 days following surgery. Conclusions: BMSC-coated bioactive sutures with GDF-8 do not appear to affect in vivo rabbit tendon healing within the first week following repair despite an increased presence of quantifiable RNA level of collagen. GDF-8’s treatment efficacy of the early tendon repair remains to be defined.

When individual neurons in a circuit contain multiple neuropeptides, these peptides can target different sets of follower neurons. This endows the circuit with a certain degree of flexibility. Here we identified a novel family of peptides, the Aplysia SPTR-Gene Family-Derived peptides (apSPTR-GF-DPs). We demonstrated apSPTR-GF-DPs, particularly apSPTR-GF-DP2, are expressed in the Aplysia CNS using immunohistochemistry and MALDI-TOF MS. Furthermore, apSPTR-GF-DP2 is present in single projection neurons, e.g., in the cerebral-buccal interneuron-12 (CBI-12). Previous studies have demonstrated that CBI-12 contains two other peptides, FCAP/CP2. In addition, CBI-12 and CP2 promote shortening of the protraction phase of motor programs. Here, we demonstrate that FCAP shortens protraction. Moreover, we show that apSPTR-GF-DP2 also shortens protraction. Surprisingly, apSPTR-GF-DP2 does not increase the excitability of retraction interneuron B64. B64 terminates protraction and is modulated by FCAP/CP2 and CBI-12. Instead, we show that apSPTR-GF-DP2 and CBI-12 increase B20 excitability and B20 activity can shorten protraction. Taken together, these data indicate that different CBI-12 peptides target different sets of pattern-generating interneurons to exert similar modulatory actions. These findings provide the first definitive evidence for SPTR-GF's role in modulation of feeding, and a form of molecular degeneracy by multiple peptide cotransmitters in single identified neurons.

Background: To investigate the effect of myostatin (GDF-8) stimulation of bone marrow derived mesenchymal stem cells (BMSCs) on tenogenesis in the setting of tendon repair. GDF-8 has demonstrated the ability to augment tenogenesis and we sought to identify if this effect could lead to the focused differentiation of pluripotential stem cells down a tenocyte lineage ex vivo.Methods: Cadaveric upper limb flexor tendons were harvested, decellularized and divided into 1 cm segments. Sutures seeded with stem cells were passed through tendon segments to simulate repair. The repaired tendons were then cultured either with or without myostatin for 3, 5, and 7 days. The experiment was also repeated with non-decellularized tendons for a total of 4 groups. The tendons were then evaluated for the expression of scleraxis and tenomodulin, two biomarkers for tendon.Results: Myostatin stimulation led to an increase in expression of tenomodulin and scleraxis at 5 and 7 days in both the decellularized and non-decellularized tendons. Myostatin increased the differentiation of BMSCs into tenocytes and/or led to the upregulation of tenomodulin and scleraxis production by the native tenocytes present within the non-decellularized tendons.Conclusions: The addition of myostatin to BMSCs leads to tenocyte differentiation as evidenced by the expression of tenocyte biomarkers, scleraxis and tenomodulin. This effect is maintained in an ex vivo tendon repair model suggestive that these cells survive the passage through tendon tissue and remain metabolically active.

MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

Background A component of primary innate defense of the nasal mucosa against inhaled pathogens includes continuous, low-level release of hydrogen peroxide (H2O2) into luminal secretions. Epidemiologically, an association exists between poor air quality and increased prevalence of sinonasal disease. To understand the effects of particulate matter (PM) in nasal mucosa, we studied the release of H2O2 and interleukin 8 (IL-8) after PM exposure. Methods Human nasal specimens were collected from surgery and cultured in serum-free growth medium. Cell integrity and recovery during culture was monitored by lactate dehydrogenase (LDH) release into the medium. Cultures were exposed to PM for 24 hours in the presence/absence of diphenyleneiodonium sulfate (DPI; a nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitor). Luminex cytokine and Amplex-Red H2O2 assays were performed. Results LDH levels dropped rapidly within 2 days, indicative of stabilization and cell recovery after harvest. All cultures released H2O2 into the medium. Exposure to PM (20 μg/cm2) increased H2O2 levels significantly (94.6 ± 7.7 nM) compared to untreated controls (55.8 ± 4.0 nM; p = 0.001). PM-induced H2O2 production was partially inhibited by DPI (80.1 ± 3.8nM), indicating that cellular NADPH oxidase may be a primary source of H2O2 production. Exposure to PM increased IL-8 levels in a dose-dependent fashion (control = 2301 ± 412 MFI; 20 μg/cm2 = 5002 ± 1327 MFI; 40 μg/cm2 = 8219 ± 1090 MFI; p = 0.022). Conclusion PM increases the quantity of H2O2 released by nasal epithelial cells, indicating that PM can contribute to oxidative stress in part by activating a normal cellular defense mechanism. Exposure to PM resulted in elevated IL-8 levels and mucin production in explants. Efforts to reduce airborne PM may lead to reduced H2O2 and mucin production in sinonasal epithelium.

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a–mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a–Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a−/− mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a−/− phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a−/− mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.

TGW is an important agronomic trait that influences the yield and milling quality of wheat. In this paper, after having investigated the phenotype of TGW from 114 ITMI recombinant inbred genealogies in 4 years in Qaidam Basin, a typical plateau oasis agricultural area, and having combined 1410 molecular markers, 7 major QTL loci of the thousand gain weight were screened out with the aid of QTL network software. These loci included qTgw1B (42.6 cm), qTgw2A(77.9 cm), qTgw2D1 (25.4 cm), qTgw2D2 (51.8 cm), qTgw6A1 (56.1 cm), qTgw6A2 (62.2 cm) and qTgw7A(75.7 cm) with their genetic contribution rates between 3.29% - 19.36%. There were two epistatic effect loci2A-2D and2A-6B with their genetic contribution rates as 2.3% and 5.3% respectively. The quantitative genetic locus positioning of thousand hexaploid wheat grain weight in Qaidam Basin can assist us in better understanding the genetic regulatory network formed by TGW, and can also provide a theoretical basis for improving thousand wheat grain weight in this ecological area.

A Gram-stain-positive, aerobic bacterial strain, designated SL15-1T, was isolated from desert soil which was sampled from the rhizosphere of Ammopiptanthus mongolicus, Hangjin Banner, Ordos, Inner Mongolia, northern China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SL15-1T was clustered with Kribbella strains, sharing the highest similarity of 16S rRNA gene sequence (96.97 %) with Kribbella sandramycini DSM 15626T. Strain SL15-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unknown phospholipid, an unknown lipid and two unknown aminophospholipids as the major polar lipids. MK-9(H4) was the predominant menaquinone, while anteiso-C15 : 0, iso-C16 : 0, C17 : 1ω8c and iso-C14 : 0 were the major cellular fatty acids. Its genomic DNA G+C content was 65.3 mol%. The results of physiological and biochemical tests allowed the discrimination of strain SL15-1T from its phylogenetic relatives. Kribbella deserti sp. nov. is therefore proposed with strain SL15-1T (=CGMCC 1.15906T=KCTC 39825T) as the type strain.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-β (TGF-β) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

Background New epithelial cells are generated through the proliferation and differentiation of resident progenitor cells in the nasal cavity. In several upper airway diseases, such as cystic fibrosis and chronic rhinosinusitis, self-renewing progenitor cells may be functionally defective, or compromised in their ability, to regenerate cells that maintain normal mucociliary clearance. Herein, we describe our early work to define and characterize a rare population of human nasal epithelial putative progenitors. Methods Single-cell suspensions of human ethmoid sinus tissues were prepared following endoscopic sinus surgery. Cell surface antibodies were analyzed as candidate markers for detecting progenitor cells. A panel of antibodies, including epithelial cell adhesion molecule (EpCAM, epithelial cells), CD45 (hematopoietic cells), nerve growth factor receptor (NGFR/CD271), intercellular adhesion molecule-1 (ICAM1/CD54), and integrin-α6 (ITGA6/CD49f) were used to resolve epithelial progenitor candidates by high-dimensional flow cytometry and the gating technique of fluorescence minus one (FMO) controls. Results A rare population of approximately 0.06% of total ethmoid cells was discriminated as EpCAM−CD45−NGFR+ICAM1+ by surface markers. Use of ITGA6 was excluded based on FMO control analysis. This lineage-negative population was purified to 99% homogeneity by cell sorting and analyzed by immunofluorescence microscopy. Sorted cells were subsequently confirmed to uniformly express the transcription factor p63. Upon in vitro culture, lineage-negative clonal cells were confirmed to spontaneously differentiate into epithelial lineage-positive cells. Conclusion Using the NGFR and ICAM1 cellular coordinates, we have identified a promising population of native human nasal epithelial progenitor cells that require more formal investigation for their role in upper airway regeneration.

Background The airway epithelium generates reactive oxygen species (ROS) as a first line of defense. Dual oxidases (DUOX1 and DUOX2) are the H2O2-producing isoforms of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family in the airway epithelium. The purpose of this study was to explore the molecular expression, function, and regulation of DUOXs in chronic rhinosinusitis (CRS).

Severe forms of chronic rhinosinusitis (CRS), a common upper airway inflammatory disorder, are associated with nasal polyps (NPs). NP disease is ameliorated by glucocorticoid (GC) treatment, whose cellular effects are poorly understood. We therefore assessed the influence of GC therapy on NPs in CRS patients, focusing on regulatory T (Treg) cells. Treg cell populations were analyzed by flow cytometry in NPs and control tissues from GC-treated CRS patients and controls. After GC exposure, selective expansion of Treg cells was seen within NPs, and not blood or adjacent ethmoid tissues. To confirm direct GC effects, NPs from the same patients were biopsied prior to, and following, 1week of oral GC exposure. Direct expansion of Tregs into the same NP bed was detected in 4/4 CRS patients following GC exposure. Treg cell spikes into NPs were secondary to cellular recruitment given limited Ki67 expression within these regulatory cells. Chemokine gene expression profiling identified several chemokines, notably CCL4, induced within NPs upon GC treatment. Neutralization of chemokine receptor/ligand interactions using CCR4 small molecule antagonists reduced Treg migration towards GC-treated NPs in an ex vivo migration assay. Our findings suggest that the common use of GCs in the treatment of NP disease leads to recruitment of Treg cells from peripheral sites into NP tissues, which may be critical to the anti-inflammatory effect of GCs. Mechanistically Treg expansion appears to be conferred, in part, by chemokine receptor/ligand interactions induced following corticosteroid therapy.

Dopamine (DA) and enkephalin (ENK) release from the tuberoinfundibular dopaminergic neurons (TIDA) into the hypophysial portal circulation is fundamentally different under non-lactating and lactating conditions. The aim of this experiment was to compare the effect of a brief interruption then resumption of suckling on the temporal program of tyrosine hydroxylase (TH; rate-limiting enzyme of dopamine synthesis) and ENK regulation in dams. On post partum day 10, pups were removed for a 4-h period from a group of the dams then returned for 4- and 24-h periods. It was examined whether such a brief interruption of suckling provokes full up-regulation of TH and down-regulation of ENK, and whether reinitiation of suckling limits the extent to which TH up- and ENK down-regulate. At the end of experiment, the animals were decapitated. In situ hybridization was used to examine the expression of TH and ENK mRNA in the arcuate nucleus where TIDA neurons reside. The results showed that, on one hand, the removal of pups induced TH up-regulation, on the other hand, ENK expression also increased 8 h after removal of pups and then started to slowly decline. In dams whose sucklings were reinitiated both TH and ENK mRNAs were up-regulated at least for a day. ENK expression responded more slowly to the removal of pups than expression of TH, and after reinitiation of suckling, the temporal program of regulation of both TH and ENK expressions ran parallel in the first 24 h.

Erianthus arundinaceus is an important, closely related genus of Saccharum officinarum L. It is therefore important to understand how the chromosomes are transmitted when it hybridizes with sugarcane. The hybrids and backcross progenies of S. officinarum and E. arundinaceus and their parents were used for Karyotype analysis and to study the law of chromosome transmission. The results showed that the somatic chromosome number of both of the E. arundinaceus Hainan92-105 and Hainan92-77 were 2n = 60 = 60sm, belonging to type 1A, and the BC1 YC01-21 was 2n = 104 = 100m + 4sm, belonging to type 2C. The other six tested clones belonged to type 2B. The both F1s YC96-66 and YC96-40 that originated from Badila (2n = 80 = 70m + 10sm) with E. Arundinaceus were 2n = 70 = 68m + 2sm, which suggests an n + n transmission. The cross between YC96-66 (female parent) and CP84-1198 (male parent, 2n = 120 = 114m + 6sm) also followed the same genetic law and the somatic chromosome number of their progeny, YC01-3 (2n = 105 = 95m + 10sm). The cross derived from YC96-40 (female) and CP84-1198 (male), YC01-21 had 2n = 104 = 100m + 4sm chromosomes, following the same genetic law of n + n. However, YC01-36 had 2n = 132 = 130m + 2sm chromosomes, which suggests a 2n + n chromosome transmission. It can be inferred that the inheritance of chromosomes was very complex in the BC1. The difference in chromosome number between clones was as high as 28. This could be explained by the 2n + n transmission of chromosomes. In addition, as there was not be a regular number of haploids, this phenomenon is termed as disequilibrium hybridization.

Loss of appetite occurs in the cecal ligation and puncture (CLP) model of sepsis in conjunction with the activation of central neural stress pathways. Neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus is upregulated by several stressors and is stimulatory to feeding. To examine the response of NPY messenger RNA in the arcuate nucleus to sepsis, we used biotinylated RNA probes and a quantitative non-isotopic in situ hybridization approach in cryo-preserved sections from rats made septic by CLP. The mRNA in arcuate neurons was upregulated from the first day after CLP. By the afternoon of the third day through the morning of the fourth day, the average grey level of NPY mRNA clusters was 30% greater after CLP than after sham surgery (P < 0.05), and the integrated optical density based on both the grey level and the amount of area with detectable mRNA was 60% greater after CLP than after sham surgery (P < 0.03). Both the average grey level and area with detectable staining were positively correlated to plasma ACTH (r = 0.953 and r = 0.917, respectively, n = 10 and P < 0.01 in each case). Thus sepsis increases the expression of the mRNA for NPY in the arcuate nucleus in proportion to the magnitude of the stress response. However, the suppression of feeding behavior in the CLP model suggests that sepsis activates additional mechanisms that negate the orexigenic contribution of the neuronal increase in NPY mRNA.

Continued smoking causes tumor progression and resistance to therapy in lung cancer. Carcinogens possess the ability to block apoptosis, and thus may induce development of cancers and resistance to therapy. Tobacco carcinogens have been studied widely; however, little is known about the agents that inhibit apoptosis, such as nicotine. We determine whether mitochondrial signaling mediates antiapoptotic effects of nicotine in lung cancer. A549 cells were exposed to nicotine (1 muM) followed by cisplatin (35 muM) plus etoposide (20 muM) for 24 hours. We found that nicotine prevented chemotherapy-induced apoptosis, improved cell survival, and caused modest increases in DNA synthesis. Inhibition of mitogen-activated protein kinase (MAPK) and Akt prevented the antiapoptotic effects of nicotine and decreased chemotherapy-induced apoptosis. Small interfering RNA MAPK kinase-1 blocked antiapoptotic effects of nicotine, whereas small interfering RNA MAPK kinase-2 blocked chemotherapy-induced apoptosis. Nicotine prevented chemotherapy-induced reduction in mitochondrial membrane potential and caspase-9 activation. Antiapoptotic effects of nicotine were blocked by mitochondrial anion channel inhibitor, 4,4'diisothiocyanatostilbene-2,2'disulfonic acid. Chemotherapy enhanced translocation of proapoptotic Bax to the mitochondria, whereas nicotine blocked these effects. Nicotine up-regulated Akt-mediated antiapoptotic X-linked inhibitor of apoptosis protein and phosphorylated proapoptotic Bcl2-antagonist of cell death. The A549-rho0 cells, which lack mitochondrial DNA, demonstrated partial resistance to chemotherapy-induced apoptosis, but blocked the antiapoptotic effects of nicotine. Accordingly, we provide evidence that nicotine modulates mitochondrial signaling and inhibits chemotherapy-induced apoptosis in lung cancer. The mitochondrial regulation of nicotine imposes an important mechanism that can critically impair the treatment of lung cancer, because many cancer-therapeutic agents induce apoptosis via the mitochondrial death pathway. Strategies aimed at understanding nicotine-mediated signaling may facilitate the development of improved therapies in lung cancer.

Chronic rapid eye movement (paradoxical) sleep deprivation (REM-SD) of rats leads to two conspicuous pathologies: hyperphagia coincident with body weight loss, prompted by elevated metabolism. Our goals were to test the hypotheses that 1) as a stressor, REM-SD would increase CRH gene expression in the hypothalamus and that 2) to account for hyperphagia, hypothalamic gene expression of the orexigen neuropeptide Y (NPY) would increase, but expression of the anorexigen proopiomelanocortin (POMC) would decrease. Enforcement of REM-SD of adult male rats for 20 d with the platform (flowerpot) method led to progressive hyperphagia, increasing to approximately 300% of baseline; body weight steadily declined by approximately 25%. Consistent with changes in food intake patterns, NPY expression rapidly increased in the hypothalamic arcuate nucleus by d 5 of REM-SD, peaking at d 20; by contrast, POMC expression decreased progressively during REM-SD. CRH expression was increased by d 5, both in mRNA and ability to detect neuronal perikaryal staining in paraventricular nucleus with immunocytochemistry, and it remained elevated thereafter with modest declines. Taken together, these data indicate that changes in hypothalamic neuropeptides regulating food intake are altered in a manner consistent with the hyperphagia seen with REM-SD. Changes in CRH, although indicative of REM-SD as a stressor, suggest that the anorexigenic actions of CRH are ineffective (or disabled). Furthermore, changes in NPY and POMC agree with current models of food intake behavior, but they are opposite to their acute effects on peripheral energy metabolism and thermogenesis.

In rodents, females but not males, in response to escalating levels of estrogen, express a luteinizing hormone (LH) surge that is prompted by a surge in luteinizing hormone-releasing hormone (LHRH). It cannot take place if estrogen-sensitive afferents located in the anteroventral periventricular nucleus (AVPV) are either absent or disabled. Males appear to lack the ability to exhibit an LH surge, but it is unclear what level of the CNS contributes to this dimorphic response. This study was conducted to determine whether estrogen followed by progesterone treatment (E + P) of gonadectomized males evokes Fos activation in LHRH and AVPV neurons as it does in females. The results indicated that, consistent with the males' inability to express an LH surge in response to E + P treatment, LHRH and AVPV neurons in males failed to show increased Fos activation. Examination of neuron nuclear antigen (NeuN, a neuron-specific marker), estrogen receptor (ERα) and progesterone receptor (PR) neurons in AVPV neurons indicated that, while essentially all the neurons of the caudal AVPV in males and females are steroid responsive, the male possessed half the number of steroid responsive neurons within the caudal AVPV (where activation of Fos is maximal in females) compared to the female. Together, these data indicate that the male lacks a substantial population of steroid receptive AVPV neurons and is unable to respond to the presence of E and P and activate either AVPV or LHRH neurons.

1. The current experiment was conducted to investigate the effect of zinc sulphate (ZnSO4) and zinc methionine (Zn-Met) on growth and their effect on plasma growth hormone (GH) concentration, growth hormone receptor (GHR) and insulin-like growth factor I (IGF-I) mRNA expression in mice. 2. Ninety male KunMing (KM) mice were randomly divided into three treatments. The control group was fed on a basal diet containing 11.67 mg/kg of zinc. The ZnSO4 group and Zn-Met group were fed on the diets supplemented with ZnSO4 or Zn-Met at 30 mg/kg (containing zinc of 40.05 and 40.75 mg/kg, respectively). The mice were offered the test diets for 10 days. Weight gains and food intake were measured at the end of the experiment, zinc contents in liver and serum were determined using atomic absorption spectrophotometry; GH was determined by radioimmunoassay, the levels of GHR and IGF-I mRNA were determined with reverse transcript polymerase chain reaction. 3. Both ZnSO4 and Zn-Met enhanced weight gain and food intake in the mice, Zn-Met improved the growth and food intake more effectively than ZnSO4 did (P0.05). The both forms of zinc had no effect on GH and the level of GHR mRNA expression (P0.05) and they up-regulated the expression of IGF-I mRNA (P0.05). As compared to ZnSO4, Zn-Met enhanced the level of IGF-I mRNA significantly (P0.05). 4. Both ZnSO4 and Zn-Met had no effect on plasma GH and the expression of GHR mRNA, but they enhanced the expression of IGF-I mRNA. Zinc methionine enhanced the weight gain and up-regulated IGF-I mRNA expression more effectively than ZnSO4.

Immunohistochemical techniques offer specificity as well as flexibility for visualizing antigens. Their use with freely floating sections provides a high signal-to-noise ratio and has become a gold standard for brain and a number of other tissues. Yet this approach initially suffered from inability to keep the antigenicity in tissue sections and required immediate processing of all cut sections. Use of sucrose solutions enabled storage at refrigerator temperatures for a few days but longer-term storage was risky and either bacterial/fungal growth or evaporation of the storage solution compromised the integrity of the tissue. Our discovery 25 years ago that tissue sections can be stored for many years at -20 degrees C in an anti-freeze cryoprotectant solution with no loss of antigenicity solved this problem and has become widely used. More recently the utility of tissue stored for many years in anti-freeze cryoprotectant was pushed to new levels by testing new non-radioactive in situ hybridization (ISH) techniques that are based on modern immunocytochemistry. This review touches upon these advances in immunocytochemical technology using examples from neuroscience applications.

Inherited predisposition to thrombosis contributes to the initiation and progression of coronary artery disease (CAD). The present study was designed to explore the relationship between genetic variation of coagulation factor V and occurrence of CAD. A total of 141 unrelated patients with CAD and 175 healthy controls were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) for variation detection in all 25 exons of the factor V gene. Among the study subjects, 55 CAD patients and 73 controls were evaluated at random for response to activated protein C (APC) by Coatest APC resistance test. Polymorphisms in exon 4, 10, 13 and 16 of factor V gene were documented [642G → T(S156), 1628 → A(R485K), 4070A → G(H1299R) and 5380G → A(V1736M), respectively]. The study also identified a novel polymorphism 327A → G in exon 2 which did not alter the amino acid residue. Leiden mutation (R506Q) was not detected in any of our 316 subjects. Among the five polymorphisms, the allele frequency of 1628G → A was significantly different between the CAD patients and the controls (0.36 vs. 0.21, p < 0.05). Subjects homozygous or heterozygous for the A allele of 1628G → A polymorphism had lower normalized APC ratios than those with the GG genotype in the CAD group (1.16 ± 0.13 and 1.18 ± 0.23 vs. 1.36 ± 0.33, p < 0.05) and in the controls, indicating that A 1628 allele was associated with a poor response to APC. We conclude that the 1628G → A (R485K) polymorphism of factor V is associated with a poor response to APC and increased risk for CAD.

Progesterone is capable of facilitating or blocking the luteinizing hormone (LH) surge, depending on the timing of its administration. However, the precise targets of progesterone's actions are unknown. Since recent studies described the presence of a periventricular preoptic area (pePOA) neuron population afferent to LH-releasing hormone (LHRH) neurons that is co-activated to express c-Fos with LHRH neurons at the time of the LH surge, the present study was designed to determine if the pePOA neurons contain progesterone receptors (PRs) and whether progesterone inhibition is manifested by a failure of LHRH and pePOA neurons to become activated at the time of an LH surge. For progesterone facilitation, a group of immature rats each received a silastic capsule (1.57 mm i.d., 3.18 mm o.d., 1.5 cm long) containing estradiol-17beta (E2) in peanut oil (150 microg/ml) at 09.00 h on postnatal day 28 followed 24 h later by a progesterone implant (crystalline, 1.57 mm i.d., 3.18 mm o.d., 1.5 cm long). For progesterone inhibition, a second group of rats received the estrogen capsule and a progesterone capsule (3.35 mm i.d., 4.65 mm o.d., 3.0 cm long) together at 09.00 h on day 28, and 24 h later received only a blank capsule. On the afternoon of postnatal day 29, all animals were anesthetized and perfused for localization of c-Fos and LHRH, PRs alone, or c-Fos and PRs. The present studies determined that following a progesterone-inhibition paradigm, along with blockade of the LH surge, both activation of LHRH and pePOA neurons was low or absent. Staining of PRs in progesterone-facilitated and progesterone-inhibited rats indicated that the pePOA neurons contained PRs in similar patterns. Double labeling of c-Fos and PRs in progesterone-facilitated rats indicated that nearly all the c-Fos-positive neurons of the pePOA (80 +/- 4.2%) co-expressed PRs; in progesterone-inhibited rats, only 32 +/- 12% of few c-Fos-positive neurons also contained PRs. In no instance were LHRH neurons found to contain PRs. Taken together, these data suggest that both progesterone facilitation and inhibition likely involve direct actions of progesterone on the pePOA neurons, and are consistent with a role for the pePOA neurons in transducing steroid effects on LHRH neurons.

FMR1 premutation (PM) alleles have 55–200 CGG·CCG-repeats in their 5′ UTR. PM carriers are at risk of fragile X–associated tremor and ataxia syndrome (FXTAS). Females are also at risk for FX primary ovarian insufficiency (FXPOI). PM pathology is generally attributed to deleterious properties of transcripts with long CGG-tracts. For FXPOI, hormone changes suggest a reduced residual follicle pool. Whether this is due to a smaller than normal original follicle pool or an increased rate of follicle depletion is unclear. A FX-PM mouse the authors generated with 130 CGG·CCG-repeats in the endogenous Fmr1 gene recapitulates features of FXTAS. Here the authors demonstrate that the gross development of the ovary and the establishment of the primordial follicle pool is normal in these mice. However, these animals show a faster loss of follicles of all follicle classes, suggesting that the problem is intrinsic to the ovary. In addition, many oocytes show aberrant nuclear accumulation of FMRP and elevated levels of ubiquitination. Furthermore, PM follicles are smaller and have fewer granulosa cells (GCs) than normal. Thus, these animals have ovarian abnormalities involving both the oocytes and GCs that may shed light on the molecular basis of FXPOI in humans.

This manuscript describes the use of staining of Fos-related antigens (FRAs) as markers for changes in neuronal activity. The model system consisted of the tuberoinfundibular dopamine (TIDA) neurons located in the arcuate nucleus of the hypothalamus. Under normal conditions, these neurons are devoid of c-Fos staining even though the neurons are tonically active and can express FRAs. During specific neuroendocrine states the neurons undergo changes in activity, as described by other studies. At times when the activity is relatively high as in pregnancy and during proestrus, approximately 50%-60% of the TIDA neurons expressed FRA immunoreactivity. Moreover changes over the course of proestrus paralleled known shifts in TIDA activity (declining as the day progressed). At times when TIDA activity is suppressed, such as during lactation, FRA staining in TIDA neurons was markedly reduced or absent. Upon removal of the suckling stimulus, FRA staining rose to reach peak expression 12-24 h after pup removal (without coordinate induction of c-Fos). These data suggest that FRA staining can serve as a useful marker of activity in the TIDA neurons which permits not only assessment of stimulated activity but also suppressed function in the neurons. A cautionary note in using this approach along with acquisition of serial blood samples for hormone measurement is that surgical procedures for monitoring plasma hormone levels are associated with strong long-lived induction of FRAs (and c-Fos) in many neurons (including the TIDA neurons) that can confound interpretation of FRA staining.

Objective: Accumulating studies show the role of microRNAs in multiple human cancers. However, little is known about the role of microRNAs in nasopharyngeal cancers (NPC). Given the role of microRNAs in survival and death pathways in cancers, we hypothesized that the microRNAs may regulate the effects of cancer therapeutic agents.Method: Tumor specific MicroRNA expressions were studied in vitro in primary human NC cells (CCL-30) treated with statins. The effects on cell survival, cell cycle, and apoptosis were examined.Results: Recently, we showed the promising antitumor effects of statins in NPC via HMG Co-A pathway. Our new study examines the novel role of microRNAs in mediating the effects of statin in the inhibition of survival in NPC. Statins induced apoptosis and decreased survival of NPC. The regulation of tumor suppressor Let-7a was enhanced in cells treated with statins associated with decrease in the cell viability and proliferation, and increase in apoptosis in these cells. Further, we found th...

Gonadotropin-releasing hormone (GnRH) neurons represent the final common output of signals from the brain that regulates reproductive function. A wide range of environmental factors impact GnRH neuron activity including disease, stress, nutrition, and seasonal cues, as well as gonadal steroid hormones. The CNS response is thought to be mediated, at least in part, through intermediate signaling molecules that affect GnRH neuronal activity.In vitro, GnRH neuronal cell lines respond to a variety of ligands that activate the Jak (Janus-activated kinase)/STAT (signal transducers and activators of transcription) intracellular signaling pathway. To determine its biological function in reproduction, we used Cre (cAMP response element)/LoxP technology to generate GnRH neuron-specific Jak2 conditional knock-out (Jak2 G−/−) mice. GnRH mRNA levels were reduced in Jak2 G−/−mice when compared with controls, while the number of GnRH neurons was equivalent, indicating a reduction in GnRH gene expression. Secretion of GnRH is also reduced as basal serum luteinizing hormone (LH) levels were significantly lower in female Jak2 G−/−mice while the pituitary responded normally to exogenous GnRH. Preovulatory LH surge levels were blunted in Jak2 G−/−mice, which was correlated with reduced GnRH neuronal activation as assessed by c-Fos. However, the activation of GnRH neurons following release from estrogen-negative feedback is retained. Female Jak2 G−/−mice exhibited significantly delayed puberty and first estrus, abnormal estrous cyclicity, and impaired fertility. These results demonstrate an essential role for Jak2 signaling in GnRH neurons for normal reproductive development and fertility in female mice.

We tested the working hypothesis that Fos will identify the critical population of kisspeptin neurons that accompanies the LHRH surge using a synchronized follicular phase model in intact cycling ewes. The model generates an LH surge that starts within a defined 2-h window in a 20-d synchronized cycle. With a modified push-pull cannula in vivo LHRH release from the median eminence was sampled in luteal phase ewes, ewes undergoing an LH surge for 2–4 h, and postsurge animals whose LH surge peaked 10–12 h earlier. In vivo release of LHRH was lower in the luteal and follicular phases than in animals undergoing an LH surge (P < 0.01); it fell to presurge levels after the LH surge. Ewes killed 2–4 h after the surge started, expressed Fos in a large portion of preoptic area (POA) kisspeptin (53.90 ± 4.69%, P < 0.01) and LHRH neurons (48.20 ± 4.49%, P < 0.0001) compared with animals euthanized at any of the other times tested (under

Tandem repeat expansion is responsible for the Repeat Expansion Diseases, a group of human genetic disorders that includes Fragile X syndrome (FXS). FXS results from expansion of a premutation (PM) allele having 55-200 CGG.CCG-repeats in the 5' UTR of the FMR1 gene. The mechanism of expansion is unknown. We have treated FX PM mice with potassium bromate (KBrO(3)), a potent DNA oxidizing agent. We then monitored the germline and somatic expansion frequency in the progeny of these animals. We show here that KBrO(3) increased both the level of 8-oxoG in the oocytes of treated animals and the germline expansion frequency. Our data thus suggest that oxidative damage may be a factor that could affect expansion risk in humans.

When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but will not necessarily work at the same primary antibody concentrations used for less sensitive reactions. Recommendations of optimal titers are often not accurate and are not usually accompanied by information on the method used to test those antibodies or the specifics of the assay. When the staining does not work, the investigators do not know how to determine if the antiserum is bad, the tissue is bad, or the method is inappropriate for their staining. This unit describes detailed procedures for determining optimal staining and applying that information to three common immunofluorescence methods. Lastly, a formula is provided for converting among the different methods. © 2016 by John WileySons, Inc.

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2′disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.

The G protein-coupled receptor Gpr54 and its ligand metastin (derived from the Kiss1 gene product kisspeptin) are key gatekeepers of sexual maturation. Gpr54 knockout mice demonstrate hypogonadotropic hypogonadism, but until recently, the phenotype of Kiss1 knockout mice was unknown. This report describes the reproductive phenotypes of mice carrying targeted deletions of Kiss1 or Gpr54 on the same genetic background. Both Kiss1 and Gpr54 knockout mice are viable but infertile and have abnormal sexual maturation; the majority of males lack preputial separation, and females have delayed vaginal opening and absence of estrous cycling. Kiss1 and Gpr54 knockout males have significantly smaller testes compared with controls. Gpr54 knockout females have smaller ovaries and uteri than wild-type females. However, Kiss1 knockout females demonstrate two distinct phenotypes: half have markedly reduced gonadal weights similar to those of Gpr54 knockout mice, whereas half exhibit persistent vaginal cornification and have gonadal weights comparable with those of wild-type females. FSH levels in both Kiss1 and Gpr54 knockout males and females are significantly lower than in controls. When injected with mouse metastin 43–52, a Gpr54 agonist, Gpr54 knockout mice fail to increase gonadotropins, whereas Kiss1 knockout mice respond with increased gonadotropin levels. In summary, both Kiss1 and Gpr54 knockout mice have abnormal sexual maturation consistent with hypogonadotropic hypogonadism, although Kiss1 knockout mice appear to be less severely affected than their receptor counterparts. Kiss1 knockout females demonstrate a bimodal phenotypic variability, with some animals having higher gonadal weight, larger vaginal opening, and persistent vaginal cornification.

Cohabitation of sexually nai;ve male and female prairie voles (Microtus ochrogaster) triggers a cascade of physiological changes that result in the formation of stable pair bonds. In the present study we used the expression of c-Fos protein to identify CNS regions activated during initial social contact in heterosexual, male/male and female/female pairs. Sexually naive males and females were randomly assigned to one of five groups: control- no cohabitation, or cohabitation for 1 h with an unrelated opposite sex, an unrelated same sex, an unfamiliar same sex sibling, or removal for 24 h and then repaired with the familiar sibling. Heterosexual pairing resulted in significant increases in c-Fos immunoreactivity (IR) in the posterodorsal and posteroventral medial amygdala (MeA), bed nucleus of the stria terminalis, medial preoptic nucleus, ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMN-VL) in males and females, and the periventricular nucleus of the thalamus in males only. c-Fos IR during intrasexual cohabitation varied with the relationship of the experimental animal to the stimulus animal. Males cohabited with an unfamiliar unrelated male expressed significantly more c-Fos IR in the central amygdala (CeA). While females cohabited with an unfamiliar female (related or unrelated) also displayed increased c-Fos IR in the CeA, this increase was accompanied by an increase in c-Fos IR in the VMN-VL and MeA. The results from this study suggest that early neuronal activation associated with heterosexual cohabitation is similar in both sexes, while neuronal activation is sexually dimorphic in response to intrasexual cohabitation.

The middle-age decline in reproductive function is manifested by reduced LHRH release, resulting in a decreased magnitude and delay of onset of the LH surge. Earlier studies suggested that the reductions in LHRH neural activation in middle-aged rats resulted from deficits in the afferent drive to the LHRH neurons. One critical afferent to the LHRH neurons lies in the anteroventral periventricular preoptic area (AVPv) nucleus. The neurons of the medial AVPv are synchronously activated to express Fos with LHRH neurons at the time of an LH surge in young adult animals. The present study examined whether, in middle age, reductions in the activation of AVPv neurons accompany the reduction in Fos activation in LHRH neurons. Young (3- to 4-month-old) and middle-aged (10- to 12-month-old) spontaneously cycling and ovariectomized steroid-replaced rats were killed during peak and early descending phase of the LH surge, and their brains were examined for Fos in LHRH and AVPv neurons. Young animals had a characteristic increase in Fos expression in both LHRH and AVPv neurons. In middle-aged rats, the proportion of LHRH neurons expressing Fos at the time of an LH surge was reduced by approximately 50%, irrespective of whether surges were spontaneous or induced by exogenous steroids. A similar reduction in the number of Fos+ cells (by approximately 50%) was noted in the medial AVPv. Linear regression analysis of the relationship between the extent of Fos activation in LHRH and AVPv neurons revealed a strong positive correlation (r2 = 0.66; P < 0.01), suggesting that changes in the AVPv’s drive to LHRH neurons underlie the decrease in LHRH activity in middle age. A second series of experiments examined whether decreased input from the AVPv could account for reduced Fos activation in LHRH neurons seen in middle-aged animals. When the medial AVPv was lesioned, LHRH neurons failed to express Fos on the side ipsilateral to the lesion. Animals with lesioned medial AVPv also had significantly lower LH values than animals with an intact medial AVPv. Taken together, these data suggest that a principal deficit in middle-aged rats is the ability of the medial AVPv to stimulate LHRH neurons.

Tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons is suppressed during lactation but rebounds upon pup removal. A time course of TH mRNA changes after pup removal revealed three phases: (1) a nuclear phase (evident 1.5 hours after pup removal, maximal at 3 hours) with TH mRNA appearing in 1 or 2 nuclear loci with little or no change in cytoplasmic mRNA; (2) a cytoplasmic phase (noted 6 hours after pup removal, peaked 12‐24 hours) with a significant increase in total TH mRNA levels mainly in the cytoplasm; and (3) a stabilization phase (24 ‐ 48 hours after pup removal) when nuclear signals were low and cytoplasmic RNA showed a slight decline with extension of RNA clusters into the cell dendrites. In rats whose pups could suckle only on one side, TH was up-regulated only on the side contralateral to nipple blockade. These data indicate that after suckling terminates, TH up-regulation is evident at 1.5 hours, but 6 hours is needed before the cells transport sufficient mRNA into the cytoplasm. The rapid signaling of TH upregulation stems from the fact that the TIDA neurons respond to neural signals from termination of suckling. J. Comp. Neurol. 438:423‐ 432, 2001. © 2001 Wiley-Liss, Inc. Indexing terms: in situ hybridization; nuclear transcription sites; hypothalamus; dopamine; neuroendocrine; prolactin; gene expression

Earlier studies demonstrated coactivation of the periventricular preoptic area (pePOA) with LHRH neurons at the time of an induced or spontaneous LH surge, suggesting that the pePOA might regulate LHRH neurons. To investigate this hypothesis, studies were conducted to determine the temporal pattern of pePOA Fos activation during the rat estrous cycle and establish the connections of the pePOA neurons with LHRH neurons. Fos activation within LHRH and pePOA neurons showed the same temporal pattern. Both were absent during diestrous I, diestrous II, and the morning of proestrus. Fos was induced in the pePOA and LHRH neurons beginning on the afternoon of proestrus (4 h before lights off), with a decline 8 h later on proestrous evening. Tract-tracing studies then established the relationship between LHRH and pePOA neurons. Retrograde labeling with fluorogold determined that a portion of the Fos-positive pePOA neurons present at the time of the LH surge sent a projection to regions that contain LHRH cells. Anterograde tracer (neurobiotin) injections established that the pePOA neurons sent axons to the LHRH cells. Taken together, these data indicate that the pePOA provides direct input to LHRH neurons that is likely to stimulate LHRH neurons at the time of the LH surge.

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

AIM: To analyse the changes of foreign plasmid copies in different tissues after uptake via gastrointestinal tract and to evaluate the possibility of foreign plasmid integrating on the host genome. METHODS: Samples including lung, kidney, spleen, mesenteric lymph node, thymus, gonads, feces, duodenum, large intestine, blood and liver were obtained 1, 3, 6, 24, and 48 h and 3, 6 wk after oral administration of 200 μg plasmid pcDNA3s. PCR technique was used to detect the distribution and kinetics of plasmid in different tissues. Genomic DNA was assayed for integrated plasmid by PCR after purification of high-molecular-weight genomic DNA away from free plasmid by using gel electrophoresis. RESULTS: Plasmid could be detected in almost all tissues 1 h after oral administration and the copies of plasmid in tissues changed with time. Foreign plasmid could be detected only in kidney and blood at sixth week time. Foreign plasmid mainly as fragment survived in vivo.