Murine vibrissae cultured in serum-free medium reinitiate anagen.
Aberrant regulation of the hair cycle has been implicated in human boldness (Reviewed in (Nakamura et al, 2001; Paus and Cotsarelis, 1999)). Although several modulators of mammalian hair follicle cycle have been recently described, discovery of effective treatment for boldness has suffered from the absence of a reliable in vitro culture systems in which the hair cycle can be assessed easily and inexpensively (Stenn and Paus, 2001). Several laboratories have established serum-free culture system where vibrissa can grow and differentiate in vitro (Supplemental Table 1, (Jindo et al, 1993; Robinson et al, 1997; Yano et al, 2001). Although rat vibrissae cultured for 23 days were reported to share histological similarities with catagen or pro-anagen (telogen) stage follicles (Philpott and Kealey, 2000), these follicles did not show any progress beyond pro-anagen phase nor did they produce a new hair shaft (Philpott and Kealey, 2000). In this report, we demonstrate that a simple modification permits murine vibrissae in the current in vitro culture system to reinitiate anagen. This will accelerate the development of screening systems aimed at modifying the hair cycle. 
 
To establish a modified in vitro vibrissa culture system, anagen-stage vibrissae were carefully isolated from 14-day-old mice and the tip of the vibrissa shafts were anchored in a stripe of sterilized silicone grease placed on the culture dish through a 3 ml syringe. The plate was then filled with serum-free medium (Figure 1a, Supplemental Material and Method). Out of 86 cultured vibrissae collected from three pups, 81 vibrissae (94%) showed measurable shaft growth (Table 1, Figure 1b). Two of 81 vibrissae were lost, and 17 developed abnormal (kinked) fiber and were omitted from growth measurement. Of the remaining 62 vibrissae, straight shafts were produced by all at a rate of 0.3–0.5mm/day for the first three days. While some follicles maintained this growth rate through the fifth day of culture (Figure 1b and 1c and data not shown), others began a gradual decline in growth rate (Figure 1b and 1c, ​,2a2a and data not shown). As previously reported (Jindo et al, 1993; Robinson et al, 1997), growth rates of all follicles slowed down considerably after 5 days in culture, indicating independence from culture conditions (Supplemental Table 1). 
 
 
 
Figure 1 
 
Vibrissae produce hair shafts and reinitiate anagen in serum-free culture medium 
 
 
 
 
 
Figure 2 
 
Histological and immunohistochemical characteristics of new anagen follicles 
 
 
 
 
 
Table 1 
 
Summary of the in vitro vibrissa culture 
 
 
 
Hair shaft elongation rate decreases and eventually stops when follicles enter catagen/telogen phase in vivo (Alonso and Fuchs, 2006). Several days after cessation of hair shaft growth, cultured vibrissae follicles continuously changed their morphology, at times visible through their collagen capsules . Strikingly, dissection and removal of collagen capsules from 21-day-cultured vibrissae revealed that all vibrissae produced a second shaft (Figures 1d and 2a–h, arrows; Figure S1), readily distinguishable from the original shaft whose proximal end formed club-end indicative of a completed catagen (Figures 1 and ​and22 closed arrowheads). Histological and immunohistochemical analyses using AE13, a marker for cortex/cuticle specific keratins, confirmed the formation of new hair shafts (Figures 2c–f and S1). The new shafts were produced in proximal bulbs that appeared smaller in size than the original ones before culture. Since the original hair growth stopped within the first 5 days of culture, and since a new shaft emerges from a secondary bulb, we concluded that vibrissa follicles from CD1 mice reinitiated anagen phase by regenerating hair bulbs . Staining for alkaline phosphatase activity identified an intact (but small) dermal papilla within the new bulb ; Young and Oliver, 1976). Moreover, the hair matrix cells were positive for Ki-67, a proliferation marker, even after 13 days in culture (Figure 2h), indicating that the formation of new shaft was the result of active proliferation and differentiation of matrix cells in cultured vibrissae. Similar cycling patterns were also observed with vibrissae from 14-day-old C57BL/6 mice , indicating that hair cycle in vitro is independent of mouse strains. 
 
To better visualize the hair cycle process, we next performed vibrissae culture after careful removal of their collagen capsules . Within 2 days, the proximal end of the vibrissa keratinized, forming a club hair, at which time dermal papilla structures were no longer discernable (Figure 2i and data not shown). A day later, however, a bud-like structure emerged from the proximal part of the bulb in eight of ten cultured vibrissae. Of these, one vibrissa continued growing outwards, eventually forming a new bulb producing a vibrissa thinner than the club hair . Strikingly, we observed that this vibrissa appeared to enter a third anagen, producing a new bud from the second bulb . Collectively, the data presented here indicate that in our in vitro culture system, vibrissa follicles enter a second anagen phase and it may be possible for a few to enter a third cycle in culture. 
 
We are not certain which modification in our culture system permits vibrissae to cycle. We do not think this is caused by differences in media composition, mouse strain, or frequency of media change. Our attempt to grow vibrissae at the air-fluid interface as in (Jindo et al, 1993) showed that one of five vibrissae formed a new hair shaft (Figure S3, Arrows), indicating that hair cycle is not strictly dependent on culture media or on the frequency of media change. However, we observed that on a solid substrate most vibrissae gradually lost their hair matrix possibly due to a pressure caused by friction generated while extending a hair shaft on a solid surface or in the absence of sebum (Sundberg et al, 2000) . Although not proven, we speculate that the critical modification was our method of immobilizing the hair shaft in silicon grease that provided friction-free environment; anchoring the tip of shafts may have allowed gravity to facilitate the separation of club hairs from the hair matrix and that supports new hair shaft formation. In addition, the smaller size of mouse vibrissae, in comparison with human or rat vibrissae, could also be a contributing factor in the anagen reinitiation in vitro As this culture system is the first to support reinitiation of anagen in culture dish, we hope this improvement will expedite the testing and development of hair cycle modifying pharmaceuticals.

title: Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes
abstract: Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfβ and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfβ ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfβ receptors, consistent with paracrine Tgfβ signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.

title: Chicoric acid: chemistry, distribution, and production
abstract: Though chicoric acid was first identified in 1958, it was largely ignored until recent popular media coverage cited potential health beneficial properties from consuming food and dietary supplements containing this compound. To date, plants from at least 63 genera and species have been found to contain chicoric acid, and while the compound is used as a processing quality indicator, it may also have useful health benefits. This review of chicoric acid summarizes research findings and highlights gaps in research knowledge for investigators, industry stakeholders, and consumers alike. Additionally, chicoric acid identification, and quantification methods, biosynthesis, processing improvements to increase chicoric acid retention, and potential areas for future research are discussed.

title: Heritable Imprinting Defect Caused by Epigenetic Abnormalities in Mouse Spermatogonial Stem Cells1
abstract: Male germ cells undergo dynamic epigenetic reprogramming during fetal development, eventually establishing spermatogonial stem cells (SSCs) that can convert into pluripotent stem cells. However, little is known about the developmental potential of fetal germ cells and how they mature into SSCs. We developed a culture system for fetal germ cells that proliferate for long periods of time. Male germ cells from embryos 12.5–18.5 days postcoitum could expand by glial cell line-derived neurotrophic factor, a self-renewal factor for SSCs. These cells did not form teratomas, but repopulated seminiferous tubules and produced spermatogenesis, exhibiting spermatogonia potential. However, the offspring from cultured cells showed growth abnormalities and were defective in genomic imprinting. The imprinting defect persisted in both the male and female germlines for at least four generations. Moreover, germ cells in the offspring showed abnormal histone modifications and DNA methylation patterns. These results indicate that fetal germ cells have a limited ability to become pluripotent cells and lose the ability to undergo epigenetic reprogramming by in vitro culture.

title: Development of a conditional gene expression system using a zearalenone-inducible promoter for the ascomycete fungus Gibberella zeae.
abstract: The ascomycete fungus Gibberella zeae is an important plant pathogen that causes fusarium head blight on small grains. Molecular studies of this fungus have been performed extensively to uncover the biological mechanisms related to pathogenicity, toxin production, and sexual reproduction. Molecular methods, such as targeted gene deletion, gene overexpression, and gene fusion to green fluorescent protein (GFP), are relatively easy to perform with this fungus; however, conditional expression systems have not been developed. The purpose of this study was to identify a promoter that could be induced by zearalenone (ZEA) for the development of a conditional expression system in G. zeae. Through microarray analysis, we isolated one zearalenone response gene (ZEAR) whose expression was increased more than 50 times after ZEA treatment. Northern blot analysis showed that the ZEAR transcript dramatically increased after 1 h of ZEA treatment. To determine the utility of the ZEAR promoter, called Pzear, in a conditional expression system, we transformed a Pzear::GFP fusion construct into G. zeae. Our data showed a ZEA concentration-dependent increase in GFP expression. We also replaced the promoter of G. zeae metE (GzmetE), an essential gene for methionine biosynthesis, with the Pzear promoter. The growth of the Pzear-GzmetE mutant on minimal medium was dependent on the ZEA concentration supplemented in the medium and showed that GzMetE expression was induced by ZEA. This study is the first report of an inducible promoter in G. zeae. Our system will be useful for the characterization of essential gene functions in this fungus through differential and ZEA-dependent gene expression. In addition, the Pzear promoter may be applicable as a biosensor for the detection of ZEA contamination in agricultural products.

title: Gallbladder damage control: compromised procedure for compromised patients
abstract: BackgroundThe objective of this study was to analyze a population-based database for (1) recent 9-year trends in utilization of partial cholecystectomy (PC), laparoscopic PC, and trocar cholecystostomy (TC), (2) demographics, associated diagnoses, and hospital characteristics, and (3) relevant inpatient outcomes.MethodsRetrospective cohort analysis of the Nationwide Inpatient Sample (NIS) files from 2000 to 2008 was performed. For the purposes of the study, gallbladder damage control was defined as PC, laparoscopic PC, and TC.ResultsA national estimate of 10,872 gallbladder damage control cases was obtained. Procedures performed included PC (47.8 %), laparoscopic PC (27.2 %), TC (25.3 %), and intraoperative cholangiogram (IOC) (19.7 %). A total of 1,479 (13.6 %) postoperative complications were identified, including pulmonary complications (4.3 %), hemorrhage/hematoma/seroma (3.4 %), and accidental puncture or laceration during procedure (3.3 %). Common bile duct injury occurred in 3.3 % overall. Hospital types included nonteaching (82.1 %) and urban (67.8 %), with regional variations of 42.1 % from the South and 45.2 % from the West. Inpatient outcomes included mean length of stay of 11.4 (0.16 SEM) days, mean total hospital charge of $71,296.69 ($1,106.03 SEM), 7.4 % mortality, and 16.8 % discharge to skilled nursing facility. Multivariate logistic regression analysis identified independent risk variables for common bile duct injury: teaching hospitals (OR = 1.517, CI = 1.155–1.991, P = 0.003). IOC (OR = 2.030, CI = 1.590–2.591, P < 0.001) was a commonly associated procedure in the setting of common bile duct injury.ConclusionVarious circumstances may require gallbladder damage control with PC and TC. Postoperative complications and common bile duct injury remain significantly high despite limited resection, and the teaching status of the hospital is associated with CBD injury. High morbidity and mortality of gallbladder damage control may reflect both the compromised nature of the procedures and multiple comorbidities.

title: Spectrum measurement of fast optical signal of neural activity in brain tissue and its theoretical origin
abstract: Functional brain imaging technologies have enabled recent advances in understanding human brain function. In particular, a noninvasive and fast optical technique will become one of the most promising tools for future studies. As a fundamental ex vivo study for the development of such a technique, this paper demonstrates the spectral measurement of fast optical changes associated with neural activity in bulk brain tissue. For this purpose, a high-speed confocal near-infrared spectrometer was built and used. The results verified that observed transient optical responses (tORs) are associated with neural activity by showing that the following: tORs were concurrent and correlated with local field potentials (LFPs); tORs disappeared following tetrodotoxin application; and tORs were reproducibly observed across preparations. In addition, the amplitude of tORs was statistically significantly larger in longer wavelengths (approximately 1200 nm). The time course of tORs, however, is quite different from that of LFPs. Since this difference implies that tORs may originate not directly from the electric potential variation but from other neurophysiological events accompanying excitation, this paper tested the hypothesis that tORs are attributed to transient cellular volume changes (tCVCs). With no previous dynamic equation to elucidate such different temporal dynamics of the optical responses, a novel mathematical neuron model to describe tCVCs was developed. This neuron model, along with finite-difference time-domain simulations, showed that tCVCs and tORs were similar in the temporal dynamics, order of magnitude, response direction, and detectability in the bulk tissue; thus supporting the hypothesis.