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Introduction Fat embolism syndrome FES is characterized by

Fat embolism syndrome (FES) is characterized by the onset of respiratory, neurological, cutaneous, and hematologic manifestations and is thought to be related to intravascular embolization of fat. FES has been associated with bone marrow necrosis in patients with hemoglobinopathies; however, it is often not considered in the primary differential diagnosis of acute complications with hemoglobin SC disease.

Report of a case
The patient was a 36-year-old female with hemoglobin SC disease. Her past medical history was significant for migraines, bipolar disorder, tobacco abuse, and asthma. She presented to an outside hospital with headache, nausea, and photophobia. Four days later she started becoming hypoxic and was subsequently intubated. She became febrile, and her hemoglobin trended down from 12 to 10g/dL with associated thrombocytopenia (platelets of 37×109/L) and elevation in her total bilirubin. Upon transfer to our institution, laboratory studies revealed hematocrit of 20%, platelets of 27×109/L, lactate of 6.3mmol/L, and acidosis with bicarbonate of 13mmol/L. Ultrasound showed no acute bleeding via Focused Assessment with Sonography in Trauma (FAST) protocol and a limited echocardiogram showed an ejection fraction of 70% and no wall motion abnormalities. Her condition further deteriorated and she twice went into pulseless electrical activity and expired within 24h of arrival.

Autopsy findings
Post-mortem examination revealed a bilateral, diffuse involvement of the pulmonary vasculature by numerous small fat emboli seen on Oil Red O stains (Figs. 1 and 2). In addition, there were bilateral pleural effusions with left lung adhesions to the A-769662 and pleural cavity wall; the right lower lobe of the lung showed a focal hemorrhagic lesion. The spleen showed splenomegaly and siderofibrotic plaques (Fig. 3) consistent with the patient’s history of hemoglobin SC disease. Leptomeningeal blood vessels and microvasculature in the gray and white matter showed congestion and circular clearings within the blood-filled vessels, suspicious for fat emboli, which were confirmed using Oil Red O stain (Figs. 4 and 5). The gastrointestinal tract showed multiple scattered hemorrhagic and infarct regions in the small bowel and peritoneal sanguineous fluid. The bone marrow showed erythroid hyperplasia.

This patient’s pulmonary and central nervous system symptoms were due to the widespread capillary embolization of fat in the lungs and brain. This is a known complication of sickle disease with marrow infarction. There are two theories – mechanical theory and metabolic theory- to explain the pathogenesis of fat embolism. The mechanical theory postulates direct embolization of fat liberated from the marrow of injured bones. The fat is driven out by an increased intramedullary pressure, transmits via the draining veins and lodges the pulmonary capillaries. The metabolic theory suggests that emboli arise in the plasma by agglutination of pre-existing tiny chylomicrons in physiological suspension. The agglutination possibly due to some biochemical change initiated by tissue damage, such as increased C-reactive protein.
In sickle cell disorders the fat emboli are thought to arise from bone marrow necrosis. Bone marrow necrosis was described for the first time in literature, in 1941, in a patient with sickle cell disease who died of cerebral infarction. Later reports suggest bone marrow necrosis is a relatively common event in the painful crises of the sickle cell disorders and usually has a full recovery after the end of the hemolytic crisis. Mechanical obstruction of the microcirculation is proposed as the mechanism for bone marrow necrosis in sickle cell disease. Aggregates of deformed sickle cells occlude the bone marrow capillaries during crisis.
Patients with hemoglobin SC disease have higher incidence of fat embolism syndrome than patients with hemoglobin SS disease. The reason has been suggested due to the higher hematocrit level in SC disease leads to larger number of sickled cells per unit volume and greater increase in blood viscosity, which predispose the patient to bone marrow necrosis. The high incidence in hemoglobin SC disease is also attributed to the increased amount of fat cells in bone marrow of SC disease patients.

br Conclusion br Acknowledgments br Introduction



A. baumannii is a leading cause of nosocomial bacteremia, severe pneumonia, meningitis, infections of the urinary tract, bloodstream and other parts of the body. The emergence of sharply increasing rate of Multidrug-resistant A. Baumannii (MDRAB) infection among critically ill, hospitalized patients, and subsequent epidemics, is a pressing problem in many hospitals worldwide. One of the reasons is that the bacteria can live up to 5months on undisturbed surfaces depending on tolerance to desiccation, and multidrug resistance. Emergence of MDRAB is due to antibiotic-selective pressure and its treatment has been challenging because of extensive antimicrobial drug resistance.
Colistin, often used as one of the last-resort RO4929097 manufacturer for multidrug-resistant Pseudomonas aeruginosa, and Acinetobacter spp., is also effective against new Delhi metallo-β-Lactamase multidrug-resistant Enterobacteriaceae. Colistin, produced by certain strains of Bacillus polymyxa var. colistinus, is a polycationic antibiotic of cyclic polypeptides. Colistin interacts with the bacterial outer membrane by displacing bacterial counter cationic in the lipopolysaccharide and solubilizes the membrane in an aqueous environment.
Resistance to colistin is not common, but A. baumannii resistance to colistin has been reported before. Recently, there has been an alarming trend with regards to the emergence of colistin-resistant A. baumannii. The pressing goal of this research, therefore, was to study the genetic characteristics and mechanisms of colistin-resistance in MDR A. baumannii.
In several gram-negative bacteria, such as Salmonella enterica and Pseudomonas aeruginosa, the mechanisms of colistin resistance include modifications, absence or complete loss of the lipid A of the outer membrane lipopolysaccharide (LPS), which reduce or abolish initial charge-based interaction with colistin. The PmrA/B system is involved in downregulating the LPS core and lipid A regions, and the lpxA/D/C gene mutations are involved in modifying biosynthesis of lipid A in resistant strains. However, the mechanism of colistin resistance in A. baumannii is currently unclear. Lipopolysaccharides (LPS) form the major component of the outer membrane of gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and providing an effective hydrophobic barrier to a variety of aggressive conditions, including antibiotics. RO4929097 manufacturer Lipid A is a crucially important component of LPS, engaging in phosphorylated glucosamine disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains anchor the LPS into the bacterial membrane. Expression of pmrA/B gene -regulating system is involved in the lipid A biosynthesis amount. The lpxA, lpxC and lpxD genes determine mainly the first three steps in the pathway of lipid A biosynthesis, and the hydrophobic anchor of lipopolysaccharide (LPS). Our recent work indicated that pmrA and pmrB gene overexpression could down-regulate lipid A biosynthesis resulting in the increase of colistin resistance. Mutations of lpxA, lpxC and lpxD, may play a role in the development of colistin resistance in clinical A. Baumannii isolates. These strains have lost their capability of producing lipid A and therefore LPS.
Understanding the mechanism of resistance to colistin may guide the development of new therapies that are imperative for MDR A. baumannii treatment. It is, therefore, very important to discover alternative therapies coupled with the development of a clinical antibiotic resistant monitor testing and the prevention of transmission of MDR A. baumannii infections.

Materials and methods


In our present collection of colistin-resistant isolates of A. baumannii, we also found 3 colistin-resistant-dependent strains (Col-D). Colistin-resistance as well as colistin-dependence have been documented previously in other species. E-test showed that MICs of colistin for these 3 Col-D strains were over 256µg/ml and MICs of Colistin for other Col-R strains ranged from 8 to over 256µg/ml respectively.

br Theoretical framework br Design research projects br Conclusion br

Theoretical framework

Design research projects



Banham and modernity
In architectural context, modernity is defined more often by examples than by theories. Consequently, there are idiosyncrasies in its movement. Functionalism stands out from such idiosyncrasies (Sharp, 2002: 4; Benevolo, 1977: 436; Dean and Zevi, 1983; Birkert, 1994: 3). Reyner Banham is one of many architectural historians who unveils and supports functionalism. He recalls the 1850s decree of Horatio Greenough: “Beauty is the promise of function” (Whiteley, 2003: 295). In this sense, the history of architecture is neither a record of stylistic development nor a chronicle of most celebrated buildings. With this position, the notion of architecture at that time is in question. Banham quotes this to challenge for a reformulation of history of architecture in dealing with contemporary circumstances.
What is modernism in architecture? Studies on modernism in architecture have been presented in several publications. Rykwert (1983) argues that the essential idea of modernism had been posed since the 18th century in French Academy. Accordingly, the French intellectuals and architects ranging from Claude Perrault to Nicholas Louis-Durand, have already put the Vitruvian Greco-Roman architectural doctrine in question; here, modernism is understood in the broadest sense of the words as the awareness of the contemporary world in the context of practice and technique. For Banham, the question on modernity is neither rhetorical nor prophetic, either in content or in tone. He takes the question seriously, which leads to an intricate investigation into the possibility of technology. History and somatostatin receptor of architecture in Banham\’s mind are indispensably inquisitive rather than a dry, dispassionate, and uncritical narrative. As far as the somatostatin receptor history and theory of modern architecture is concerned, no one is idiosyncratically able to talk and write about it without going through Banham\’s positions and expositions.
As one of the most profound theorists and historians on functionalism in the Age of Machine Aesthetics, the relation of Banham to modernism in architecture is seemingly neither a “father and son” relationship nor a “subject and object” binary, but acquired immunodeficiency syndrome (AIDS) might be properly said as a co-existential pair of the 20th century architectural history. To certain extent, Banham is more than just an observer and a witness of historical movement of modern architecture. Properly speaking and regarding his rigorous scholarship, Banham is probably one of the best references of knowledge, power and subject of modernity in architectural history. As an editor of Architectural Review (1952), Banham is the man in action in polemics and debates on contemporary architecture in Western world.
The approach to Banham\’s works in this study is considerably hermeneutical by which Banham\’s concepts and its modern contextuality will be necessarily dismantled and unfolded for their intrinsic and explicit meanings and significances. By nature, the study is to make modernism a case based upon Banham\’s passion in the dynamic relationship between technological innovations and artistic endeavours that happens and makes a history for the presence of architecture. This study will emphasize its analysis in an explorative way that enables one to see the interplay between power, knowledge, and subject in Western industrial and capitalist cultures. The goal of this study is to uncover and to unfold Banham\’s vision on history of architecture as the immediate future of comprehensive ecosystems, instead of dated works in classified styles by names of architects (Banham, 2009: xxxiv). In order to achieve this goal, this study will handle three categories of architectural presence: function, technology, and aesthetics. These threefold presence will be studied with respect to Banham\’s thoughts, positions, commentaries, notes, and unspoken messages.

NVP-AUY922 cost The most significant characteristic of active

The most significant characteristic of active learning is student involvement: students are actively engaged in individual or group activities during the class session, these may include reading, discussing, commenting, and exploring tasks, ideas and theories (Liebman, 1997). Rather than declamatory orator, the instructor takes on the more active role of facilitator and/or mentor and can thus provide students with immediate feedback (Bonwell, 1996). Notably, in active learning sessions students are involved in accessing higher order thinking; this simultaneously involves the analysis, synthesis, and evaluation of a wide spectrum of issues and phenomena. In the context of an active-learning university classroom, students are engaged not only in doing things but also in reflecting and thinking about what they are doing (Dean, 1996). In essence, the pedagogical literature and research findings of the past few decades demonstrate the value and validity of active learning.
Experiential learning has developed into an important paradigm based on the works of John Dewey, Jean Piaget, and David Kolb. They argued that a practical, hands-on experience should be an integral component of any teaching/learning process; this rationale must apply to classroom settings. These arguments vividly echo the famous saying of the Chinese philosopher Confucius, who more than two thousand years ago promoted experiential learning: ‘Tell me and I will forget. Show me and I may remember. Involve me and I will understand.’ Therefore, experiential learning, unlike learning in which the learner only reads about, hears about, talks about, or writes about these realities but never comes in contact with as part of the learning process, is first hand learning in which the learner is directly in touch with the realities being studied (Keeton and Tate, 78,1978; Salama, 2015).
In the context of the discipline of architecture and urban design, there are educators who mistakenly equate experiential learning only with ‘off campus’ or ‘non-classroom’ learning, not conceiving how it could be very effectively applied to the classroom setting. For example, instead of providing students with dull lectures about theories of architecture and the work of famous architects, a class in the history of architecture or urban design, or a class in design theories might incorporate periods of student practice on NVP-AUY922 cost exercises and critical thinking problems (Salama, 2012b). Likewise, a class in ׳principles of architectural design׳ or in ׳human-environment interactions׳ might involve critical analysis exercises on how people perceive and comprehend the built environment. Both classes could require field visits to buildings and spaces where students are in close contact with the environment, thus enabling them to better explore aspects of culture, diversity, and people׳s behaviour, while actively being part of that environment. Hence, these mechanisms involve an experiential learning component which thus enables students to experience and explore the first-hand problems they examine or discuss in the classroom setting.
Learning through experience involves not merely observing the phenomenon being studied but also doing something with it or to it, for example testing its dynamics or applying a theory to learn more about it and/or achieve desired results. Assessment of environments as a valuable research vehicle that needs to be introduced in lecture courses; this can help establish a solid knowledge base about the built environment which will enable students to have more control over their learning, knowledge acquisition, assimilation, and utilisation in future experiences. Such an approach corresponds with John Habraken׳s call to legitimise design professions by incorporating learning about the everyday environment (Habraken, 2006).
While including assessment research and active and experiential learning as interactive learning mechanisms that enable the effective comprehension and dissection of the built environment, it is also important to involve architecture and design students in assessment processes that are conducted objectively and systematically: casual interviews or observations may only reveal what is already known, not what has been learnt and internalised. Through experiential learning, students are actively engaged; they learn about the problems and potentials of existing environments and how or whether they meet user needs, enhance and celebrate their activities, and foster desired behaviours and attitudes. Recent work by the first author reveals that although there have been several attempts to incorporate assessment research into architectural pedagogy, it would appear that most have not gone beyond individual attempts of a few committed scholars and educators (Salama, 2015). Thus, we argue that traditional teaching practices do not employ interactive learning mechanisms that effectively address the dialectic relationship between people and their environments to help students better understand and grasp the multifaceted nature of the built environment.

The Wilcoxon test was performed by using

The Wilcoxon test was performed by using SPSS software for Windows (Version 10.0, SPSS, Chicago, Illinois, USA). A p value of <0.05 was considered statistically significant.
As compared to baseline values, PdetQmax decreased in 28 patients, increased in 12, and had no change in the other 6 after TUI-BN. Overall, the mean Qmax increased (baseline vs post-TUI-BN, 5.72 ± 4.77 vs 13.7 ± 8.02 mL/second, p < 0.001), the voided volume increased (146 ± 130 vs 185 ± 109 mL, p = 0.03) and PVR decreased (214 ± 200 vs 108 ± 136 mL, p < 0.001) significantly after TUI-BN. The glucose transporter neck became wide open and had a funnel shape during voiding in all patients after TUI-BN (Fig. 1B).
Table 1 shows that the mean Qmax increased, the voided volume increased, and the PVR volume decreased significantly after TUI-BN in both the increased-Pdet and decreased-Pdet groups. PdetQmax significantly increased and decreased in the increased Pdet and decreased Pdet group after TUI-BN, respectively. The other six patients maintained low detrusor contractility and large PVR volumes after TUI-BN. Among the patients with no change in detrusor contractility after TUI-BN, four voided efficiently, with assistance of abdominal pressure, while the remaining two patients had persistent voiding difficulty. No major complications, such as bleeding, urinary incontinence, or retrograde ejaculation, were reported. PPBC improved by 2 scales after TUI-BN in 36 (78%) patients, including 22 (79%) in the decreased-Pdet group, 10 (83%) in the increased-Pdet group, and 4 (67%) with persistent detrusor underactivity.

This study showed that TUI-BN improved voiding function in most patients with primary BND, with or without high voiding pressure before surgery. Patients with BND are usually treated with alpha-blockers, and when medical therapy fails to improve urinary symptoms, surgery with TUI-BN seems to be effective, which was concretely confirmed by the results of this study. Improved bladder voiding function was achieved by TUI-BN in patients with BND and impaired detrusor contractility. Even in the patients with persistent detrusor underactivity, 67% of them voided adequately with the aid of abdominal straining.
In the past, no BND pathophysiology was definitively elucidated, but several theories were postulated. The initial theory focused on the structural change of the bladder neck, e.g., fibrosis or hyperplasia. Smooth muscle hypertrophy, fibrous changes and inflammatory changes were also reported as the possible causes of BND. Some studies suggested that BND could be caused by abnormalities of the striated urethral sphincter muscle, and this dysfunctional external sphincter extended to the bladder neck in 48% of men. Recently, the concept of sympathetic inhibition was raised and neuropeptide Y was considered as a possible neurotransmitter for bladder neck dyssynergia. In addition to sympathetic hyperactivity, decreased acetylcholine release over time was also considered as a possible cause influencing bladder contractility. Decreased acetylcholine levels in rat bladders were also found in association with partial bladder outlet obstruction. These experimental results help explain why low detrusor contractility is present in patients with primary BND.
In clinical practice, the key diagnostic criterion of BND is the characteristic appearance of a narrowed or unopened bladder neck, while elevated voiding pressure is usually, but not always, present in these cases. In our study, 12 patients with low Pdet at baseline had improved bladder voiding function after TUI-BN. This finding is quite different from previous observations, that patients with BND should have a high voiding pressure, and the decreased voiding pressure and increased urinary flow rate are always noted after TUI-BN.
During normal voiding, detrusor contraction starts following urethral sphincter relaxation and bladder neck funneling. The bladder neck and external urethral sphincter are innervated by the sympathetic nervous system. The role of sympathetic nerves on bladder filling has recently been emphasized. Alpha-adrenergic nerves are postulated to inhibit reflex activation of the detrusor muscle during bladder filling, while beta-adrenergic nerves relax the detrusor muscle. Experimental data support that the released norepinephrine exerts an inhibitory effect on detrusor function by adrenergic innervation.

Simple closure of the perforated ulcers should

Simple closure of the perforated ulcers should not be an option. During the operation, maximum attention must be paid in order not to miss any small, shallow ulcers at different locations.
One study showed that the cumulative recurrence rates after surgical treatment were 29.2% at 2 years and 47.2% at 5 years; the cumulative reoperation rates were 12.5% at 2 years and 22.2% at 5 years. Multivariate analysis identified volcano-shaped ulcers, higher C-reactive protein levels, and a history of postoperative steroid therapy as independent predictive factors for reoperation.
Fistula formation after the appendectomy is a very rare complication (0.13% was reported in one study) and usually occurs after complicated appendicitis such as gangrenous or perforated appendicitis. Of greater significance is the fact that fistulas usually develop from the ileum in cases of BD, but not from the appendiceal stump.

Congenital umbilical hernia (CUH) is a congenital malformation, not uncommon in infants, and the male-to-female ratio is roughly equal. Although sometimes quite large, most umbilical hernias in children resolve on their own without any treatment if they are asymptomatic, reducible, and do not enlarge by the age of 2–3 years. When the fascial defect is small (<1 or 2 cm), 90% of all umbilical hernias close within 3 years (85% in some reports), regardless of size. Complications of an umbilical hernia that require immediate surgical intervention are incarceration or strangulation and in extremely rare cases, rupture, when the skin over the hernia breaks open, exposing the tissue inside the hernia sac. These complications are rare as the underlying defect in the abdominal wall is larger than in the inguinal hernia of the newborn. The size of the order monensin of the herniated tissue is inversely correlated with the risk of strangulation (i.e., a narrow base is more likely to strangulate). In developing countries, patients with emergency surgical problems present late, often after complications have developed. A total of 20 cases of spontaneous rupture of pediatric umbilical hernia have been reported in the literature and we will add such a case of a 45-day-old girl with evisceration of the small intestine.

Case report
A 45-day-old girl had a CUH, but it was easily reducible. Treatment was not thought to be necessary by her parents, as they were advised to let the child have conservative management by a physician. On the day of rupture, the hernia appeared normal. It developed suddenly during crying episodes after a feed. Her mother noticed that the umbilical swelling which had been reducible before, became irreducible. Crying episodes became more frequent and the swelling enlarged and rapidly became tense, and as a result of an obstructed loop of bowel within it, the abdomen became increasingly distended. Redness appeared on the inferior aspect of the skin around the swelling. Severe vomiting ensued, and this forced the parents to ask for another medical opinion. Following intravenous support at a local hospital, the baby was referred to our institute for further management. On the way to the hospital, crying episodes caused spontaneous rupture of the thinned umbilical skin with subsequent evisceration of the loops of bowel within the defect. The infant was rushed to our department. Clinically the patient was stable except order monensin for the ruptured umbilical hernia with eviscerated ileum (Fig. 1). The surrounding tissues appeared edematous, but there was no evidence of infection or necrosis. Careful inquiry failed to elicit any other cause for the rupture except for a history of paroxysms of crying probably due to incarceration of the bowel during morning hours. There was no suggestion of trauma by the napkin-pin. An assessment of the spontaneous rupture of the umbilical hernia was made. The infant was resuscitated with intravenous fluid and nasogastric tube suction, and the eviscerated bowel was covered with saline-soaked sterile gauze. The patient was also treated with sedatives, analgesics, and antibiotics. Laboratory investigations were carried out and the patient underwent an emergency operation.

Although the bulge derives from the ectoderm a

Although the bulge derives from the ectoderm, a cell population of mesodermal origin is also present within the hair follicle tissue (Schneider et al., 2009). Mesodermal derived tsa inhibitor are located in the dermal papilla and dermal sheath and appear to regulate hair follicle development and cycling through cross-talk with the epithelium (Schneider et al., 2009). Hair follicle dermal papilla/sheath cells promote hair restoration upon transplantation (Jahoda et al., 1984; McElwee et al., 2003) and have been reported to be immunoprivileged (Reynolds et al., 1999). Previous studies showed that rodent dermal papilla/sheath cells have broad differentiation potential, similar to bone marrow derived mesenchymal stem cells (Jahoda et al., 2003; Hoogduijn et al., 2006). Notably, transplantation experiments showed that cells derived from the dermal papilla/sheath of mouse hair follicles reconstituted multiple lineages of the hematopoietic system of lethally irradiated mice, suggesting that these mesenchymal cells have very broad differentiation potential (Lako et al., 2002). Recently our group demonstrated that dermal papilla/sheath cells from human hair follicles exhibited mesenchymal stem cell (MSC) immunophenotype and differentiated to all mesenchymal lineages and therefore, they were termed human hair follicle derived mesenchymal stem cells (hHF-MSCs) (Liu et al., 2010). In addition, using a smooth muscle α-actin promoter we derived functional smooth muscle cells (SMC) from human and ovine HF-MSCs, which were used to engineer small-diameter vascular constructs exhibiting robust contractility in response to vasoactive agonists (Liu et al., 2008; Peng et al., 2011).


The hair follicle is an easily accessible organ that covers large areas of the skin and harbors distinct populations of stem cells, probably owing to its diverse developmental (ecto-mesodermal) origin (Cotsarelis et al., 1990; Morris et al., 2004; Tumbar et al., 2004; Jahoda et al., 2003; Yu et al., 2006). Previous studies showed that cells from the dermal papilla/sheath of rodent (Jahoda et al., 2003; Hoogduijn et al., 2006) and human hair follicles (Liu et al., 2010; Liu et al., 2008) displayed characteristics of mesenchymal stem cells but human HF-MSCs have not been investigated in detail. Here we addressed two questions: (i) how does in vitro culture affect the proliferation and multi-lineage differentiation potential of hHF-MSCs; and (ii) are hHF-MSCs clonally multipotent? We found that hHF-MSCs are highly proliferative cells that can be maintained in culture for ~45 population doublings before they begin to show signs of cellular senescence. Gene expression profiling and functional assays demonstrated that hHF-MSCs can differentiate to myogenic, osteogenic, adipogenic and chondrogenic lineages. Interestingly, although adipogenesis decreased significantly over time in culture, myogenesis and chondrogenesis decreased to a much lesser extent even after 11 passages, while osteogenic capacity was highest at intermediate passages. In addition, the majority of hHF-MSCs were clonally multipotent, suggesting that they may present an alternative source of easily accessible, autologous stem cells for tissue engineering and regenerative medicine.
hHF-MSCs displayed high proliferation potential over several passages. Based on the average number of population doublings per passage (2.5–3.5), it was estimated that hHF-MSCs underwent ~36 population doublings during the first 12 passages. Addition of 8–10 population doublings that took place during the initial isolation and expansion phase brings the total number between 44 and 46 population doublings. This compares favorably with previous reports estimating 13–25 population doublings for human BM-MSCs (Wagner et al., 2008), although admittedly more accurate comparison would require that both hHF-MSCs and BM-MSCs be isolated from the same donors and account for sex and age differences. Regardless, during the first 2weeks of cell isolation a single scalp hair follicle can give rise to approximately 5×104 MSCs, which then undergo ~36 additional population doublings yielding ~1015 cells. Given that the density of hair follicles in the scalp is over 200 follicles/cm2 (Barman et al., 1965) and that most of them are in the anagen phase for prolonged times, the potential of hHF-MSCs as a stem cell source for regenerative medicine is very promising.

3X FLAG tag Peptide br Author contribution The following are the supplementary data related

Author contribution
The following are the supplementary data related to this article.

We thank Catherine Bresse for her valuable assistance in the statistical analysis. This work was supported by the CIRM grant # RL1-00636-1.

The capacity of human embryonic stem 3X FLAG tag Peptide (hESCs) for self-renewal, propagation, and maintenance of the pluripotent state in vitro offers the potential to utilize hESC technology for therapy of many severe human diseases as well as cell-based assays (Klimanskaya et al., 2005; Mallon et al., 2006; Rosler et al., 2004; Thomson et al., 1998; Xu et al., 2001). However, many negative factors contribute to current inefficient culture systems for hESCs, which have limited the implementation of such a therapy. The ineffectiveness of hESC culture systems is due to (i) low plating efficiency when cells are seeded as single cells or small clumps (Androutsellis-Theotokis et al., 2006; Watanabe et al., 2007), (ii) very low recovery rates when cells are thawed after cryopreservation (Li et al., 2009), and (iii) acquired heterogeneous cellular states owing to various cellular stresses under excessive apoptotic or differentiation signals (Adewumi et al., 2007; Hartung et al., 2010). In addition, xenogeneic contaminants from any non-human feeder cells or foreign components of the culture system may also impede future clinical application (Mallon et al., 2006).
To solve these problems, we need to establish a robust and reliable system for hESC culture and assay. Standard colony-aggregated culture exhibits slow expansion and often gives rise to heterogeneous cells (Adewumi et al., 2007; Hartung et al., 2010) and frequent chromosomal abnormalities (Baker et al., 2007; Draper et al., 2004; Lefort et al., 2008; Maitra et al., 2005; Spits et al., 2008). Hence, a non-colony type culture is preferable. The use of JAK inhibitor I (JAKi) and the Rho-kinase inhibitor Y-27632 (ROCKi) has been shown to significantly improve single-cell plating efficiency in both neural stem cell and hESC cultures (Androutsellis-Theotokis et al., 2006; Chen et al., 2010; Li et al., 2009; Ohgushi et al., 2010; Pakzad et al., 2010; Watanabe et al., 2007).
Intuitively, hESCs with high single-cell plating efficiency using these small molecules could enable us to propagate the cells in a single-cell based non-colony type monolayer (NCM) culture, which would greatly improve the current culture conditions. Various defined substrates have been reported to support hESC culture as colonies under feeder- or xeno-free conditions (Klim et al., 2010; Mallon et al., 2006; Melkoumian et al., 2010; Rodin et al., 2010; Villa-Diaz et al., 2010). The use and characterization of a NCM method as an independent culture system for the maintenance of undifferentiated hESC lines under defined substrate conditions have not been reported. In this study, we report such a hESC culture system for facilitating pluripotent stem cell growth and assays.

Materials and methods

Results and discussion

The following are the supplementary data related to this article.

Disclosure of potential conflicts of interest

The NIH Stem Cell Unit is supported by NIH funds. We thank Dr. Joshua Chenoweth for the discussion, Dr. Peter Andrews for providing antibodies described in Supplemental information, Dr. Guokai Chen for the BC1 line, and Dr. Tianmin Ivy Zhang and Dr. Qi Zheng (Applied StemCell Inc.) for conducting the teratoma assays.

Glioblastomas, the most common and malignant primary brain tumor, have a dismal prognosis, despite modern refinement of diagnostic techniques and treatment strategy including surgery and radio/chemotherapy. Median survival of the patients with glioblastoma is generally about a year from the time of diagnosis and this has not significantly improved for more than three decades (DeAngelis, 2001; Stewart, 2002; Stupp et al., 2005). As a novel treatment strategy, gene therapies using the herpes simplex virus-thymidine kinase (HSVtk) gene and ganciclovir (GCV) were clinically tested (Rainov, 2000; Ram et al., 1997). However, the results were unsatisfactory partly due to limited migratory activity of the vector-producing cells derived from fibroblasts, which could not cover all of the glioma cells that widely infiltrated into the surrounding brain tissues. To improve the migratory activity of the effector cells, use of the neural stem cells and bone marrow stromal cells (BMSCs), that display extensive tropism for brain lesions including gliomas, has been introduced (Aboody et al., 2000; Benedetti et al., 2000; Li et al., 2007). In the previous study, we demonstrated that established intracranial glioma in the rat brain was successfully treated by intratumoral injection of BMSCs transduced with HSVtk gene (BMSCtk cells) followed by intraperitoneal GCV administration (Amano et al., 2009). This is mainly due to a very potent “bystander effect”, where tumor cells that are not transduced with HSVtk gene are eliminated when mixed with HSVtk-expressing cells by GCV administration (Culver et al., 1992), as well as an active tumor tracking ability of BMSCs (Lee et al., 2003; Nakamizo et al., 2005). In fact, we have demonstrated a surprisingly potent bystander effect between BMSCtk and C6 rat glioma cells (Amano et al., 2009). These observations suggest that “BMSCtk therapy” is promising as a novel clinical treatment strategy for glioblastomas.

Consistent with our in vitro data two

Consistent with our in vitro data, two recent genetic studies in the mouse provide strong in vivo evidence that Pdx1 is a bona fide transcriptional repressor. By E11.5, the pancreatic buds in Pdx1 null mutant embryos arrest and begin to regress (Ahlgren et al., 1996; Offield et al., 1996). Recently, Seymour et al. (2012) performed high-resolution quantitative immunohistochemistry on Pdx1−/− embryos and observed significant numbers of ectopic Afp+ Cy5.5 NHS ester Supplier within the dorsal pancreatic bud. This suggestion of partial conversion to the hepatic cell fate is consistent with prior work demonstrating that hepatic competence is not restricted to the region of the ventral foregut where the liver normally forms (Bossard and Zaret, 1998, 2000; Gualdi et al., 1996) and with our data that PDX1 represses AFP in vitro (Figures 4D–4F). Seymour et al. (2012) also showed that Pdx1 deficiency caused varying degrees of Sox9 downregulation and that focal loss of Sox9 in the developing pancreas led to elevated expression of hepatic markers. Our results show that in hESC-derived ePP cells PDX1 binds SOX9 between exons 2 and 3 (Table S1, part A), which suggests that SOX9 is positively regulated by PDX1, as expected for these principal regulators of the pancreatic program. Phylogenetic sequence conservation in this region of mouse and human SOX9 genes (data not shown) fits the idea that this Pdx1-Sox9 regulatory relationship is central to the pro-pancreatic gene regulatory network. Taken together, these findings indicate that early-stage Pdx1+ progenitor cells are not stably determined (“metastable”), with Pdx1 positively regulating Sox9 and actively repressing liver potential during a substantial period of early pancreas organogenesis.
In a second and very recent study, Gao et al. (2014) inactivated Pdx1 in the adult β cell using Cre-Lox methods with concurrent indelible YFP labeling of the derived Pdx1−/− cells (Gao et al., 2014). Expectedly, these mice became rapidly hyperglycemic—a result consistent with prior work (Ahlgren et al., 1998; Gannon et al., 2008)—but unexpectedly lineage-labeled Pdx1−/− cells contained glucagon and expressed MafB, a transcription factor that in the adult mouse is restricted to the islet α-cells. These authors used ChIP from mouse insulinoma cell lines to detect PDX1 binding within 1.5 kb of the MafB TSS. Taken together, these findings strongly suggest that Pdx1 directly represses MafB transcription in adult β cells. Interestingly, MafB is required for the production of both α and β cells during pancreas development, but its expression is extinguished in β cells soon after birth (Hang and Stein, 2011). Consistent with this in vivo expression kinetic, we observed MAFB levels increasing from days 0 to 17 of hESC differentiation (Figure 3B). However, MAFB transcriptional regulation is apparently independent of PDX1 at these stages, as PDX1 binding was observed a great distance from the MAFB TSS (≥150 kb) (Table S1, part A). In addition, it is important to highlight recent data showing that, in contrast to mice, MAFB persists in a subset of human adult β cells (∼9%) (Dai et al., 2012). This finding suggests that in mice Pdx1 adopts its role as a transcription repressor late in β cell ontogeny and that in humans PDX1+MAFB+ and PDX1+MAFB− represent distinct β cell subtypes.
Our findings raise an important outstanding question: what is the mechanism underlying PDX1 transcriptional repression? We speculate that one answer lies in the top-ranking motifs enriched in our ChIP-seq data—PBX1 and FOXA1/A2. Nearly 20 years ago, Pdx1 was shown to bind the HOX-cofactor Pbx1 (pre-B cell leukemia factor 1), a member of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing proteins (Peers et al., 1995). Pbx1 alternative splicing yields two isoforms differing at their C termini, the longer Pbx1a and shorter Pbx1b. Pdx1:Pbx1b complexes transcriptionally activate target genes, while Pdx1:Pbx1a forms a repressor complex through the recruitment of co-repressor proteins such as NCoR-SMRT (or HDAC) (Asahara et al., 1999; Saleh et al., 2000). PBX1A and PBX1B are both expressed during human ePP differentiation (Figure S1A; A.K.K.T. and N.R.D., unpublished data), raising the possibility of differential recruitment within the same cell to activate or repress appropriate gene targets to direct lineage choice and stabilization. Similarly, FOXA transcription factors can also recruit HDAC via the co-repressor Groucho-related protein 3 (Grg3; formally Tle3), which is highly expressed in the pancreas during embryonic development, to silence genes central to hepatic differentiation (Lam et al., 2013; Santisteban et al., 2010). Our data also show that FOXA1/A2 binding sites were significantly enriched in sequence reads from day 17 PDX1 ChIP-seq (Figure 2A), and FOXA2:PDX1 co-binding was observed frequently in nearly 2,000 loci in mouse islets (Hoffman et al., 2010). Canonical FOXA1/A2 motifs exist close to PDX1 binding in both AFP and TTR, also suggesting context-dependent functions of FOXA proteins. Finally, it is important to note that approximately 100 genes are bound by PDX1 on day 17, but their expression is significantly downregulated by microarray (comparing day 17 to day 0) (Figure 3C; Table S2, part E). Obvious candidates for direct repression among these ∼100 include FGF8, TWIST2, ETV4, and ZIC3, whose mouse orthologs are typically expressed in mesoderm or mesodermally derived tissues during embryonic development ( (Table S2, part E). Methods to address the issue of direct repression or activation of PDX1 target loci include the development of PDX1-deficient hESC lines or lines carrying inducible knockdown tools for context and time-dependent inactivation.

purchase GW788388 br Introduction Hematopoiesis is thought to

Hematopoiesis is thought to begin from stem purchase GW788388 that progress through consecutive precursor cell stages in a hierarchical fashion whereby lineage commitment precludes alternative cell differentiation. However, there has been increasing evidence of hematopoietic plasticity and cell lineage conversion, particularly during leukemogenesis (Cobaleda and Busslinger, 2008; Graf, 2008; Greaves et al., 1986; Regalo and Leutz, 2013). The transcription factors C/EBPα and C/EBPβ are potent inducers of myelomonocytic genes in heterologous cell types (Ness et al., 1993), and the experimental conversion of lymphoid cells to myeloid cells modulated by both C/EBPs has highlighted their lympho-myeloid transdifferentiation potential (Graf and Enver, 2009).
The C/EBP family members C/EBPα, C/EBPβ, C/EBPδ, and C/EBPε are expressed in myeloid cells (Cloutier et al., 2009; Scott et al., 1992). Loss-of-function studies in genetically modified mice suggested combinatorial and partially redundant functions of C/EBPs in myelopoiesis (Tsukada et al., 2011). Knockout studies showed that deletion of Cebpa has the strongest impact on myelopoiesis, resulting in an almost complete loss of neutrophils and impaired development of granulocyte-macrophage progenitor (GMP) cells (Zhang et al., 1997, 2004). However, cytokines could compensate for the lack of Cebpa by the concomitant activation of Cebpb, and genetic replacement of Cebpa with Cebpb in the Cebpa locus compensates for the Cebpa requirement in hematopoiesis and liver functions (Chen et al., 2000; Hirai et al., 2006; Jones et al., 2002). Individual deletions of C/EBPβ, δ, and ε evoke milder and gene-specific phenotypes, such as susceptibility to infections, failure of emergency granulopoiesis, impaired cytokine production, and partial granulocyte deficiency that is intensified by compound C/EBP gene deletions. For example, compound Cebpb/Cebpe deletion mutants display impaired granulopoiesis, defective macrophage functions, and a disrupted innate immune regulatory gene expression network, confirming the compensatory and redundant functions of the C/EBPs (Akagi et al., 2010; Hirai et al., 2006; Litvak et al., 2009; Tanaka et al., 1995; Yamanaka et al., 1997).
C/EBPα can stimulate the transdifferentiation of B and T cells and, together with PU.1, even fibroblasts into macrophages (Bussmann et al., 2009; Feng et al., 2008; Ness et al., 1993; Xie et al., 2004). Conversion of B cells into inflammatory-type macrophages occurs rapidly after C/EBP expression, with high efficiency and through a direct route (Bussmann et al., 2009; Di Tullio et al., 2011; Xie et al., 2004). An experimental transdifferentiation system based on an estrogen-responsive, conditional C/EBPα protein in the v-H-ras-transformed pre-B cell line HAFTL1 (Holmes et al., 1986) has served as a tool to examine the mechanistic aspects of lympho-myeloid lineage conversion, including alterations of chromatin occupancy, gene expression kinetics, non-coding RNA expression, and DNA methylation (Barneda-Zahonero et al., 2013; Di Tullio et al., 2011; Kallin et al., 2012; Krijger et al., 2016; Rodriguez-Ubreva et al., 2012, 2014; van Oevelen et al., 2015).
We recently found that structural alterations and post-translational modification sites of C/EBPβ may determine the path of transdifferentiation of primary progenitor B cells toward distinct myeloid cell fates (including granulocytes and dendritic cells), suggesting that epigenetic instructions beyond the inflammatory macrophage cell fate are encoded in the C/EBP structure and could account for cell-type specification (Stoilova et al., 2013). This observation has prompted us to compare the lineage conversion capacity of all transactivator C/EBP family members and to develop a lympho-myeloid transdifferentiation system that is amenable to targeted mouse genetics and cell-culture manipulation.
In this study, we generated murine v-Abl-immortalized B cells from wild-type and genetically altered mice to compare the lympho-myeloid transdifferentiation potential of the C/EBP family members C/EBPα, C/EBPβ, C/EBPδ, and C/EBPε. Our data showed that C/EBPβ and C/EBPε readily induce a granulocytic fate in addition to macrophage formation. Granulocytic conversion largely depended on transgene dosage. In addition, efficient transdifferentiation required endogenous Cebpa/Cebpb. Importantly, applying selective pressure on immortalized B cells expressing C/EBPβ by depriving β-mercaptoethanol resulted in the rapid extinction of B cells and massive expansion of stable myeloid cells. These myeloid progenitors displayed bipotential GMP-like properties and continuously produced macrophages and granulocytes. This process could suggest a link between C/EBP-induced lympho-myeloid lineage switch and a B cell-derived leukemic myelomonocytic GMP-like phenotype (Slamova et al., 2013).