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Salivary gland acinar cells are routinely destroyed during radiation treatment for head and neck cancer that results in a lifetime of hyposalivation and co‐morbidities. A potential regenerative strategy for replacing injured tissue is the reactivation of endogenous stem cells by targeted therapeutics. However, the identity of these cells, whether they are capable of regenerating the tissue, and the mechanisms by which they are regulated are unknown. Using in vivo and ex vivo models, in combination with genetic lineage tracing and human tissue, we discover a SOX2+ stem cell population essential to acinar cell maintenance that is capable of replenishing acini after radiation. Furthermore, we show that acinar cell replacement is nerve dependent and that addition of a muscarinic mimetic is sufficient to drive regeneration. Moreover, we show that SOX2 is diminished in irradiated human salivary gland, along with parasympathetic nerves, suggesting that tissue degeneration is due to loss of progenitors and their regulators. Thus, we establish a new paradigm that salivary glands can regenerate after genotoxic shock and do so through a SOX2 nerve‐dependent mechanism.
Salivary glands regenerate after radiation injury through SOX2‐mediated secretory acinar cell replacement as shown using genetic lineage tracing and ablation methods, in combination with in vivo and ex vivo gamma radiation‐induced damage models.
SOX2+ stem cells are essential to acinar cell replacement in the sublingual gland (SLG) during homeostasis and after radiation‐induced damage.
SOX2‐mediated acinar cell replacement is contingent on neuronal muscarinic signalling.
In the absence of nerves, a muscarinic mimetic can drive SOX2‐mediated regeneration.
SOX2 function is essential for SLG regeneration following radiation‐induced injury.
SOX2 along with parasympathetic nerves are diminished in human salivary gland biopsies following irradiation therapy and SOX2 and the acinar lineage are upregulated in response to muscarinic activation.
Therapeutic radiation continues to be a life‐saving treatment for cancer patients and is utilized for a spectrum of malignancies including those of the head and neck. Indeed, the vast majority of patients suffering head and neck cancer will receive radiotherapy in addition to chemotherapy and surgery (~60,000 new patients per year in US; Siegel et al, 2015). Although this combination treatment is highly efficacious in eliminating tumors, a severe side effect is damage and/or destruction of healthy tissue lying in the field of radiation. Such organs include the salivary glands, which exhibit tissue dysfunction even after low doses of radiation (Grundmann et al, 2009). At the higher doses routinely given to patients (60 Gy), off‐target radiation destroys saliva‐synthesizing acinar cells (Sullivan et al, 2005; Redman, 2008) and results in a lifetime of dry mouth and co‐morbidities (e.g., tooth decay, oral infections, poor wound healing (Brown et al, 1975; Dreizen et al, 1977; Dusek et al, 1996). Although there has been success with intensity modulated radiation to spare one of the three major salivary glands (parotid), the proximity of the glands to the tumor sites often prevents application of this technique, leaving 80% of head and neck cancer patients with dry mouth syndrome (Lee & Le, 2008).
As with all other organs damaged by radiation, including the lungs, heart, and bladder (Emami et al, 1991), there are few, if any, treatments available to improve or restore tissue function. Current treatment options for cancer survivors suffering radiation‐induced salivary dysfunction and degeneration focus on short‐term relief from the symptoms, but no long‐term restorative therapies are available. Regenerative strategies such as reactivating endogenous stem cells or transplanting non‐irradiated stem cells have been proposed (Lombaert et al, 2008; Ogawa et al, 2013; Pringle et al, 2016). However, these applications are curtailed by the dearth of knowledge regarding the identity of adult salivary progenitor cells that contribute to acini under homeostatic or injury conditions. Although it was recently proposed that acinar cells are derived through self‐duplication rather than from defined progenitors (Aure et al, 2015), an analysis of subpopulations of these cells for progenitor‐like activity was not performed. It also remains to be determined whether acinar cells, either through self‐duplication or through progenitor cell expansion, are capable of repopulating cells after genotoxic damage. Although a plethora of studies have utilized irradiated salivary glands as a model of degeneration (Zeilstra et al, 2000; Coppes et al, 2001, 2002), the regenerative capacity of adult salivary cells damaged by radiation has not been investigated in vivo.
How acinar cells are replaced during salivary gland homeostasis is also poorly understood. Studies in adult organs over the last 150 years have clearly shown that peripheral nerves are essential for the maintenance of organ and tissue integrity (Erb, 1868). Skeletal muscle atrophies in the absence of stimulation by motor neurons (Fu & Gordon, 1995; Batt & Bain, 2013) and epithelial organs such as fungiform taste buds (Von Vintschgau & Honigschmied, 1877), prostate (Wang et al, 1991; Lujan et al, 1998) and the salivary gland degenerate after ablation of sensory and/or autonomic nerves (Schneyer & Hall, 1967; Mandour et al, 1977; Kang et al, 2010). Although it is unclear how nerves control tissue homeostasis for these organs, studies in skin indicate sensory nerves, through sonic hedgehog secretion, promote the self‐renewal of adult epithelial stem cells and consequently the maintenance of the downstream cell lineage, that is, dome cells in the skin (Peterson et al, 2015; Xiao et al, 2015). In addition, studies in the salamander (Wallace, 1972) and embryonic salivary gland (Knox et al, 2013) suggest that peripheral nerves have the capacity to regenerate tissue via activation of multipotent stem cells, but evidence for this in the adult mammalian system is lacking.
Using a combination of mouse genetics, ex vivo cultures, and human tissue explants, we unexpectedly discover that salivary acini are capable of regenerating after radiation and do so in response to cholinergic activation through a progenitor cell‐dependent mechanism. We show that SOX2 marks the sole progenitor for the acinar lineage that can replace acinar cells during homeostasis and after radiation‐induced injury, indicating that salivary progenitors can withstand, at least in the short term, genotoxic shock. Importantly, treatment of healthy and irradiated tissue with cholinergic mimetics stimulated acinar cell replenishment. Thus, our data reveal the extensive regenerative capacity of the tissue even under genotoxic shock and suggest that targeting of SOX2+ cells might be a therapeutic approach to regenerate tissue damaged by radiation therapy.
SOX2 marks a progenitor cell that gives rise to acinar but not duct cells during salivary gland homeostasis
SOX2 has been established as a progenitor cell marker in the fetal mouse submandibular and sublingual salivary glands, but whether SOX2+ cells in the adult tissue also produce acinar and duct cells is unclear (Arnold et al, 2011; Emmerson et al, 2017). Furthermore, whether these cells are also present in adult human salivary glands is not known. We found SOX2 to be expressed by a subset of acinar cells in all three of the major adult human salivary glands [Fig 1A, submandibular gland (SMG), sublingual gland (SLG), parotid gland (PG)]. In the mouse, SOX2 protein was restricted to the adult murine SLG (absent from the SMG and PG, Figs 1B and EV1A) where it was expressed by undifferentiated aquaporin (AQP)5‐positive, mucin (MUC)19‐negative acinar cells (21 ± 4% of all AQP5+ acinar cells; Fig 1C and D). Consistent with their potential role as a progenitor cell, ~6% of SOX2+AQP5+ cells co‐expressed Ki67 (Figs 1E and EV1B) while 19 ± 4% were in the cell cycle (CyclinD1+; Fig EV1C). To determine whether SOX2+ cells contributed to acinar and duct lineages, we performed genetic lineage tracing using Sox2CreERT2 mice (Arnold et al, 2011) crossed to a Rosa26mTmG reporter strain. The Rosa26mTmG mouse is a double‐fluorescent reporter which when crossed with a Cre line expresses membrane‐targeted tandem dimer Tomato (mT) prior to Cre‐mediated excision and membrane‐targeted green fluorescent protein (mG) after excision (Muzumdar et al, 2007; Fig EV1D). As such, lineage‐traced cells will express mG. As shown in Fig 1F, SOX2+ cells self‐renew and produce differentiated acinar cells marked by AQP5 and MUC19 but not KRT8+ duct cells after 14 or 30 days (Figs 1F and EV1E). Thus, our lineage tracing results indicate that SOX2+ cells are lineage‐restricted progenitor cells that give rise to differentiated progeny, similar to what has been observed in the epidermis, intestine, and incisor (Owens & Watt, 2003; Barker, 2014; Seidel et al, 2017).
Given KIT+ cells, which reside primarily in the intercalated ducts of the SLG and SMG (Andreadis et al, 2006; Nelson et al, 2013), have previously been proposed to give rise to acinar cells in adult tissue (Lombaert et al, 2008; Nanduri et al, 2013, 2014; Pringle et al, 2016), we genetically traced these cells using the KitCreERT2 promoter crossed to the Rosa26mTmG reporter at 6 weeks of age. However, no KIT+ cell‐derived acinar cells (i.e., double positive for AQP5 and mG) were evident in either the SLG or SMG at 14 days or 6 months after induction (Fig EV1F). Instead, KIT+ cells contributed exclusively to the intercalated ducts in the SLG (as can be observed by co‐staining for the intercalated duct marker KRT8) and intercalated and larger ducts in the SMG. Thus, these data indicate that KIT+ cells are progenitors for the ductal and SOX2+ cells for the acinar lineage.
SOX2 and SOX2+ cells are essential for production of secretory acini
Our lineage tracing analysis confirmed that SOX2+ cells give rise to acinar but not duct cells. However, as we also observed the presence of Ki67+SOX2− acinar cells (~6% SOX2+Ki67+ and 16.5% SOX2‐Ki67+ cells, Fig EV1B), suggestive of an alternative progenitor cell or a transit‐amplifying cell for the acinar lineage, we investigated the requirement of SOX2 and SOX2+ cells in SLG maintenance and repair by genetically removing Sox2 in SOX2+ cells using Sox2CreERT2; Sox2fl/fl mice (Fig 2A and C) or ablating SOX2+ cells using diphtheria toxin (DTA) expressed under the control of the inducible Sox2 promoter (Sox2CreERT2;Rosa26DTA; Fig 2B and D). In the latter assay, SOX2+ cells undergo cell death in response to intracellular production of DTA. Ablation of Sox2 from SOX2+ cells or elimination of SOX2+ cells via DTA severely depleted SOX2+ and AQP5+ cells but not KRT8+ ductal cells indicating Sox2 and SOX2+ cells were necessary for maintaining functional acini (Fig 2A–D; efficiency of Sox2 or SOX2+ cell ablation is shown in Fig EV2A). In the absence of Sox2, acinar but not ductal cells exited the cell cycle, as shown by the decrease in cyclin D1 (CCND1)+ acinar cells (Fig EV2D; arrowheads indicate CCND1+ cells and dotted white lines highlight ductal cells). Furthermore, ablation of SOX2+ cells resulted in few remaining acini by 8 days (Figs 2B and D, and EV2A), as shown by large regions of the ductal network completely devoid of AQP5+ cells (ducts are marked by dashed lines or KRT8 in Fig 2B). To exclude the possibility that tissue degeneration was solely due to destabilization of the tissue rather than loss of acinar cell replacement, we examined SLG after a short‐term ablation. As shown in Appendix Fig S1, at day 4 or 5 (3 or 4 days of tamoxifen treatment), few SOX2+ cells remained in the gland of both the Sox2CreERT2; Sox2fl/fl and Sox2CreERT2;Rosa26DTA SLG (Appendix Fig S1A and B) and Sox2 transcripts were substantially reduced (Appendix Fig S1B). However, acini were present albeit disorganized and atrophic in appearance. Furthermore, we did not observe an increase in SOX2+ cells (or Sox2 transcripts), indicating that SOX2 is not ectopically expressed in acinar cells in response to tissue damage. We also determined whether alterations in tissue composition were due to reduced innervation, an essential regulator of tissue function. However, we measured similar innervation in Sox2CreERT2; Sox2fl/fl SLG to wild‐type controls and a significant increase in axon bundles in Sox2CreERT2; Rosa26DTA SLG (Fig EV2B and C). The latter finding suggests ablation of cells triggers the release of factors that promote innervation but that, even with increased innervation, regeneration is not possible without SOX2+ cells. In sum, these results indicate that SOX2+ cells, at least under the conditions tested, are the sole acinar progenitors in the SLG and that acini do not arise from the self‐duplication of fully differentiated acinar cells, as suggested previously (Aure et al, 2015). Similar to studies in the epidermis, intestine, and incisor (Owens & Watt, 2003; Barker, 2014; Seidel et al, 2017), our data also suggest the presence of a transit‐amplifying population derived from SOX2+ cells that may be involved in rapidly repopulating the acinar compartment.
Parasympathetic nerves preserve SOX2+ progenitors and promote SOX2‐mediated acinar cell replacement
Adult murine and human salivary glands atrophy after removal of parasympathetic activity. However, the effect of denervation on acinar cell replacement and progenitor cells has not been investigated (Garrett et al, 1999; Raz et al, 2013). To this end, we denervated one of the two pairs of murine SLGs by transecting the chorda tympani (Fig 3A; contralateral glands were used as internal controls). After 7 days, transcript levels of neuronal genes Tubb3, Vip, and Vacht (Fig 3B, red bars) and GFRα2+ or TUBB3+ nerves (Figs 3C and EV3C) were severely reduced, indicating successful denervation. We did not observe a concurrent loss of the cholinergic muscarinic receptors Chrm1 and Chrm3 transcripts (Fig 3B, red bars); however, it is possible that in the absence of parasympathetic innervation a compensatory mechanism may maintain Chrm1 and Chrm3 transcription. Although the SLG is predominately served by the parasympathetic branch with very little sympathetic innervation in comparison (Emmelin et al, 1965), we did observe a reduction in sympathetic innervation following chorda tympani transection (Fig EV3A). As such, although the levels of sympathetic nerves are minor, we cannot rule out that some of the effects of denervation may be due to a loss of sympathetic input.
Similar to the effect of radiation therapy on tissue structure (Sullivan et al, 2005; Redman, 2008) adult acinar cells, as well as SOX2+ progenitors, were more sensitive to the loss of innervation than ducts. Denervation resulted in reduced acinar cell size (as observed previously; Patterson et al, 1975; Fig EV3B) decreased AQP5 protein and transcript levels of the differentiated acinar cell marker Muc19 (Fig 3B and D). Interestingly, transcript and protein levels of MIST1 were unchanged following denervation (Fig 3B, E and F), suggesting that while functional markers of acinar cells are disrupted in the absence of innervation, acinar cell identity is not adversely affected. Strikingly, SOX2+ cells lose expression of Sox2 (demonstrated using the Sox2eGFP mouse) and the levels of SOX2 protein and transcript were greatly reduced (Figs 3B, C and F, and EV3C), indicating SOX2 maintenance requires innervation. To determine whether SOX2+ cells remained capable of repopulating the tissue after denervation, we performed genetic lineage tracing where Cre driven by the endogenous Sox2 promoter (Sox2CreERT2;Rosa26mTmG) was activated 3 days after denervation and traced until day 14. As shown in Fig 3G and H, acinar cell replacement by SOX2+ progenitors was significantly reduced (~50%) 14 days after transection. Similarly, in SLG in which recombination was induced before nerve transection (tamoxifen 1 day prior to transection), acinar cell replacement by SOX2+ progenitors was significantly depleted (~50%) after 14 days (Fig EV3E and F). Reduced acinar cell replacement is likely due to decreased cell proliferation rather than cell death as we measured a reduction in Ccnd1, while markers of cell death [activated caspase‐3 (CASP3+) cells in Fig EV3G or Bax, Pmaip1 (NOXA) and Bbc3 (PUMA) in Fig EV3H)] were either not observed or remained unchanged. The absence of cell death also suggests that cells that were previously positive for SOX2 continue to be present but that cholinergic innervation is essential for maintaining SOX2 expression.
To confirm that denervation preferentially affects the acinar lineage in the SLG, we also analyzed other epithelial cell lineages. KRT8+ ducts in denervated glands resembled innervated controls, as did transcript levels of the ductal genes Krt7, Krt8, and Krt19 (Fig 3B, D and F). Furthermore, KRT5+ cells, progenitors in developing SMG/SLG (Knox et al, 2010; Lombaert et al, 2013) that are maintained by parasympathetic nerves (Knox et al, 2010), were unaffected by denervation (Figs 3F and EV3D; for transcript Krt5 expression see Fig 3B). Based on these findings, we conclude that parasympathetic innervation is required for adult SLG tissue homeostasis by preferentially maintaining and replacing functional acini through regulation of SOX2 and SOX2+ cells.
To determine whether resupplying salivary gland with nerves could rescue acini and SOX2, we examined murine salivary gland 30 days after denervation. Due to the plasticity of the peripheral nervous system, murine salivary glands become reinnervated over time, as shown by the reappearance of TUBB3+ nerves 30 days after transection (Appendix Fig S2A) and re‐expression of the neuronal genes Tubb3, Vip, and Vacht (Fig 3B, blue bars; Yawo, 1987). Surprisingly, we found elevated expression of these neuronal genes at day 30 (Fig 3B), suggestive of hyperinnervation, in response to the original injury. Remarkably, upon reinnervation the levels of Sox2 and Aqp5 transcripts and SOX2 protein as well as numbers of SOX2+ and AQP5+ cells and acinar cell size returned to at or above control levels (Fig 3B and Appendix Fig S2A–C).
To ensure that SOX2+ cells of the SLG were capable of responding directly to acetylcholine produced by the parasympathetic nerves, we analyzed expression of acetylcholine/muscarinic receptors by SOX2+ cells as well as their ability to respond to muscarinic agonists in vivo. SOX2+ cells expressed both CHRM1 and CHRM3 (95 and 99%, respectively; Fig 4A and B) and short‐term treatment of wild‐type mice with the muscarinic agonist pilocarpine (delivered I.P. and sacrificed at 18 h) increased the percentage of proliferating SOX2+ cells (SOX2+Ki67+ cells; Fig 4C and D). The percentage of SOX2+ cells, however, was not significantly changed by 18 h post‐injection (Fig 4D), indicating that muscarinic activation does not induce ectopic expression of SOX2. Thus, these data support our hypothesis that parasympathetic nerves via acetylcholine muscarinic signaling maintain SOX2+ progenitors and acini and promote acinar cell replenishment.
Murine salivary glands regenerate after radiation‐induced damage via SOX2
The murine salivary gland (predominantly the SMG) has been used extensively to investigate the effects of ionizing radiation (IR) on glandular function and structure, where typical analysis is limited to degenerative responses. The C57BL/6 background has been reported to undergo loss of acinar cells and a decline in salivary flow rate after a single 10 Gy dose (Zeilstra et al, 2000; Coppes et al, 2001, 2002). However, the regenerative capacity of the tissue and whether it remains innervated after IR are unknown. To test the effect of radiation on SOX2‐mediated salivary gland regeneration, we analyzed innervation, SOX2+ cells and SOX2‐mediated acinar cell replenishment in murine SLG after a single dose of gamma‐radiation to the head and neck. Similar to previous studies in salivary glands (Avila et al, 2009), we found a 10 Gy dose induces DNA damage and cell cycle arrest, as well as reduces cell proliferation in the SLG in the first day following IR, as shown by a substantial increase in the pro‐apoptotic gene Bax and the cell cycle inhibitor Cdkn1a (p21) (Fig EV4A) and a reduction in transcript levels of the cell proliferation marker Mki67 (Fig EV4A). We then measured changes in innervation and nerve function in IR SLG by immunolabeling for TUBB3+ nerves and performing semi‐quantitative PCR (qPCR) for Tubb3 and the parasympathetic nerve‐derived neurotransmitter Vip. As shown in Fig 5A and B TUBB3+ nerves were unchanged in the IR SLG compared to non‐IR controls 1 and 3 days after radiation. However, transcript levels of Tubb3 and Vip were significantly reduced at 1 and 3 days following IR, suggesting nerve function is reduced at early stages (Fig EV4B). Similarly, transcripts for Sox2, Mist1, and Aqp5 were reduced immediately following IR but returned to control levels by day 7 post‐IR (Fig EV4B). The number of SOX2+ cells was significantly reduced at day 1 post‐IR (Fig 5C), while the number of CCND1+SOX2+ cells was significantly increased at days 3 and 7 post‐IR (Fig 5C). In order to determine whether SOX2‐mediated cell replacement was affected following IR‐induced damage, we analyzed the extent of SOX2‐mediated replenishment in Sox2CreERT2;Rosa26mTmG mice 14 days post‐IR. Strikingly, we found acinar cells were replaced by SOX2+ cells in the IR SLG (GFP+ cells) similar to the non‐IR control with the production of both SOX2+GFP+ and SOX2‐negative progeny (Fig 5D, white arrowheads indicate SOX2‐negative progeny), suggestive of the presence of a transit‐amplifying cell which is no longer SOX2+ but derived from a SOX2+ cell (i.e., lineage‐traced). Furthermore, the number of SOX2+ cells in the IR SLG was similar to controls by day 14 (Fig 5D). Thus, despite an initial loss of nerve signaling, SOX2, and acinar cell markers, the acinar compartment is capable of being replenished by SOX2+ cells after radiation‐induced damage.
Next, we tested whether the SLG could regenerate following IR injury in the absence of Sox2. As shown in Fig 5E, Sox2CreERT2; Sox2fl/fl mice that had been irradiated with a single 10 Gy dose were unable to repopulate the tissue with functional AQP5+ acini. Indeed, in the absence of Sox2, we observed a loss of AQP5+ acinar cells and disrupted tissue architecture compared to wild‐type mice at 14 days post‐IR (Fig 5E). This outcome further confirms that Sox2 is essential for SLG regeneration following radiation‐induced injury.
SOX2+ cells can replenish the irradiated salivary gland in response to cholinergic mimetics
As our data suggest cholinergic cues replenish the acinar lineage in the SLG, we determined whether SOX2+ cells can replenish acini in response to muscarinic activation in healthy and irradiated SLG using our ex vivo lineage tracing model. As shown in Fig 6A and B, there was an increase in GFP+ clones in healthy SLG tissue cultured with the acetylcholine mimetic carbachol (CCh) for 48 h. This increase in GFP+ clones was associated with an increase in cell proliferation (Ki67+ cells) with CCh treatment (Fig EV4E). In our IR model, recombination was induced in Sox2CreERT2; Rosa26mTmG mice 24 h before animals were subjected to a single dose of IR (Fig 6A). This time point was chosen as a lag time of 12–24 h has been previously reported for tamoxifen‐induced recombination of Cre lines in mice (Nakamura et al, 2006). Thus, single SOX2+ cells are labeled by 24 h after injection (Fig 6B, see 0 h panels). SLGs were collected within 1 h of radiation exposure and cultured ex vivo with or without CCh for 48 h. As shown in Fig 6B and quantified in Fig 6C, GFP+ clones were more abundant in IR SLG compared to the no IR controls, suggesting IR activated SOX2+ cells to repopulate the tissue. This was likely due to cholinergic signaling from remaining nerves as IR explants cultured with the muscarinic receptor antagonist 4‐DAMP exhibited similar acinar cell replenishment to non‐IR cultured salivary gland (Fig 6B and C). Importantly, treatment of IR explants with CCh increased GFP+ cells compared to IR alone (Figs 6B and C, and EV4C and D). Based on these data, we conclude that SOX2+ cells are capable of repopulating the IR SLG in response to muscarinic activation.
Acetylcholine/muscarinic signaling maintains SOX2 and the acinar lineage in human SG
For humans, IR irreversibly reduces parasympathetic (and increases sympathetic) innervation in SG (Knox et al, 2013; Fig EV5A; GFRA2), as well as markers of the acinar lineage (AQP3, MIST1, AMY1) but not ductal lineage (EGFR, KRT19) compared to no IR controls (Fig 7A; IR was delivered ~2 years prior to surgery, n = 7 IR and 11 no IR). Lineage markers were confirmed in human tissue (SMG was used due to availability) by immunofluorescence (Fig EV5B). Furthermore, we found transcript levels for SOX2, GFRA2, CHRM1, and CHRM3, but not tyrosine hydroxylase (TH, sympathetic marker), were also significantly downregulated or trended toward downregulation following IR (Figs 7A and EV5A), indicating parasympathetic function as well as the ability of cells to respond to acetylcholine was depleted. Thus, we hypothesized that the loss of regenerative capacity of human salivary glands following IR is due to reduced parasympathetic innervation of the SOX2+ progenitor cells, and that acetylcholine/muscarinic signaling is sufficient to maintain SOX2 expression and promote the acinar lineage. To test this hypothesis, we established a novel human explant‐murine nerve co‐culture system. In this model, non‐irradiated human SMG is dissected into < 1‐mm pieces and placed alongside an embryonic day 13 murine submandibular gland parasympathetic ganglia (contains mesenchyme) or mesenchyme only (control, no nerves). Tissues are then co‐cultured on a filter floating above serum‐free media for 7 days. Nerves migrated in and around the tissue (Fig 7B) and actively maintained tissue structure as shown by higher levels of CDH1 (E‐cadherin, gene, and protein expression) in the explants co‐cultured with nerves compared to mesenchyme alone (Fig 7C and D). We analyzed the explants for the neuroattractants neurturin (NRTN) and nerve growth factor (NGF) (Knox et al, 2013) in an attempt to ascertain what factors induced this nerve migration. However, we found no increase in expression in the presence of the ganglia compared to the mesenchyme alone (Fig 7D), suggesting that either other neuroattractants were being synthesized, that the epithelia produces these neuroattractants whether the nerves are present or not (i.e., the mesenchyme is supportive enough to maintain epithelial homeostasis) or that this phenomenon occurs earlier in culture (i.e., at an earlier stage when the nerves are just starting to envelop the epithelia) and any differences have been resolved by this later time point (day 7). Strikingly, glandular explants cultured with nerves also exhibited increased transcript levels of SOX2 (~1.8‐fold to twofold) and the acinar markers MIST1 and CD44, muscarinic receptor CHRM3 (also expressed by acinar cells; Giraldo et al, 1988) but less consistently the ductal genes KRT19, KIT, and EGFR compared to the mesenchyme only controls (Fig 7D, data normalized to mesenchyme only, n = 2 separate individuals). In addition, epithelial cell proliferation, marked by the presence of Ki67+ cells, was increased in the presence of the nerves (Figs 7C and EV5C). Together, these data suggest that parasympathetic nerves are able to maintain SOX2 expression and acinar and ductal markers in the human salivary gland.
To address whether acetylcholine/muscarinic signaling is sufficient to maintain acini and SOX2+ cells in human salivary gland, we subjected non‐irradiated human SMG or PG (both express SOX2) tissue explants to muscarinic stimulation in an ex vivo system. Culture of patient‐derived human tissue from four separate individuals (n = 4) with CCh significantly increased expression of SOX2, muscarinic receptors CHRM1 and CHRM3 [both expressed by adult acinar cells (Giraldo et al, 1988; Mei et al, 1990)], as well as AQP3, AQP5 (Gresz et al, 2001), and MIST1 within 4 h of muscarinic stimulation (Fig 7E, n = 4, individual datasets shown in Fig EV5D). While surgical denervation does not adversely affect expression of Mist1 (Fig 3B and E), muscarinic stimulation is sufficient to increase MIST1 in human cultures and suggests that although not required for acinar cell identity, acetylcholine/muscarinic signaling may act as a positive regulator of the secretory program. The variability in response between the four patient‐derived samples is likely due to biological diversity between human patients, differences in the type of gland sourced (SMG and PG used), and the age of the patient (age of donor ranges from 30 to 78 years). However, in all cases, we observed an increase in SOX2 and a number of acinar markers in the presence of CCh. Although we also measured an increase in KRT19 gene expression with CCh, the other exclusive ductal markers EGFR, KRT7, and KRT8 remained unchanged, suggesting ductal cells do not generally respond to muscarinic agonists (Fig 7E). Given nerves increased ductal genes in the human salivary gland in co‐culture, other factors may be produced by nerves to elicit changes in the ducts (compare Fig 7D and E). Thus, neuronal acetylcholine/muscarinic signaling is sufficient to promote the acinar lineage and maintain SOX2 expression in adult human salivary gland.
Our study reveals SOX2+ cells as progenitors in the adult salivary gland essential to the replenishment of acini with the unexpected capacity to repopulate the tissue after radiation‐induced damage. We further show that cholinergic nerves play a vital role in controlling SOX2‐mediated acinar cell replacement during homeostasis and that this neuronal influence can be replicated through addition of cholinergic mimetics to irradiated tissue. Thus, contrary to current dogma that murine salivary glands do not regenerate after radiation‐induced damage (Zeilstra et al, 2000; Coppes et al, 2001, 2002), these data indicate that, at least in mice, salivary glands have extensive regenerative capacity after radiation‐induced damage. Furthermore, as we find that the acinar lineage (and SOX2) in human tissue is also responsive to cholinergic mimetics, targeting SOX2+ cells and maintaining cholinergic nerves may aid in the recovery of functional salivary acini after damage by radiation therapy.
SOX2 has an essential role in organism development and regulates the homeostasis of epithelial tissues such as the stomach, trachea, and intestine (Arnold et al, 2011). Similar to the reduced homeostatic capacity of adult tracheal cells after Sox2 ablation (Que et al, 2009), we found depletion of AQP5+ acinar cells after ablation of Sox2 in SOX2+ acinar progenitors under both homeostatic and damage conditions. Intriguingly, although atrophied, we did not find increased cell death in the absence of Sox2 but substantially fewer cycling acinar cells, suggesting that upon ablation, these cells exit the cell cycle and undergo differentiation to produce dormant AQP5‐deficient acinar cells. The striking loss of acini upon ablation of SOX2+ cells further confirmed that these are the sole progenitors for the acinar lineage in the SLG and is consistent with a recent report demonstrating pancreas acinar cells are not equipotent but contain a subset of progenitors (Wollny et al, 2016). Our results contrast to a recent report by Aure and co‐workers showing that salivary acinar cells replenish through self‐duplication of mature cells rather than via progenitor differentiation (Aure et al, 2015), similar to pancreatic beta cells (Dor et al, 2004). The conclusion that tissue repopulation is due to self‐duplication of salivary acinar cells was based on the use of an inducible Mist1 promoter that is expressed by all acinar cells, including SOX2+MUC19− cells. As such, subsets of cells traced upon recombination likely include progenitors; however, no analysis was conducted to determine whether these subsets possess a stem cell signature.
As with all epithelial organs, peripheral nerves are required to maintain the structural homeostasis of rodent and human salivary gland (Schneyer & Hall, 1967; Mandour et al, 1977; Wang et al, 1991; Fu & Gordon, 1995; Lujan et al, 1998; Kang et al, 2010; Batt & Bain, 2013). During organogenesis, parasympathetic nerves maintain a progenitor population that can contribute to the tissue after radiation damage via acetylcholine/muscarinic signaling (Knox et al, 2013). Although previous denervation studies in the salivary gland indicated that the organ and acinar cells decrease in size (Schneyer & Hall, 1967; Mandour et al, 1977; Kang et al, 2010), whether innervation was required for acinar cell replacement was not known. Here we reveal that nerves directly regulate SOX2 to drive acinar cell replacement from lineage‐restricted progenitors. This outcome is consistent with the known role of SOX2 in the regulation of self‐renewal and cell fate in many other organs (Arnold et al, 2011). However, to date, only cell‐intrinsic signaling pathways including those mediated by the WNT, FGF, and EGFR families have been shown to regulate SOX2 (Hashimoto et al, 2012; Dogan et al, 2014; Rothenberg et al, 2015; Lee et al, 2016). This extrinsic nerve‐based model has the distinct advantage over cell‐intrinsic signals in that, unlike their target organs, neurons themselves are highly resistant to radiation damage (Tofilon & Fike, 2000; Wong & Van der Kogel, 2004). Whether this mechanism also regulates the maintenance of other SOX2‐expressing epithelial organs, for example, taste buds (Suzuki, 2008), prostate and seminal vesicles (Wanigasekara et al, 2004), stomach (Tatsuta et al, 1985; Zhao et al, 2014), and cornea (Ueno et al, 2012), remains to be tested. However, these results suggest cholinergic nerves may function in the regeneration of these tissues.
Previous studies have utilized the murine salivary gland as a model of radiation‐induced degeneration (Zeilstra et al, 2000; Coppes et al, 2001, 2002). These investigations have been based on the assumption that regeneration is impaired after moderate to high doses of radiation, a hypothesis supported by the reduced saliva flow measured in animals receiving radiation (Redman, 2008). However, to date, there has been no in vivo analysis of cell replacement after radiation. Our data indicate that murine acinar cells are highly regenerative, at least in the first 30 days after radiation exposure, and are capable of repopulating the acini similar to uninjured controls. It is clear, however, that this regenerative capacity cannot be sustained for the long term as degeneration/senescence in murine salivary glands occurs 3–6 months after radiation (Urek et al, 2005; Marmary et al, 2016). As such, it is likely that the regenerative capacity of SOX2+ cells does fail eventually and further analysis is required to discern the cause. It also remains to be determined whether the human salivary gland can regenerate in the days/months after therapeutic radiation and if this regenerative capacity fails in the long term due to the absence of SOX2+ cells in combination with parasympathetic nerves. Indeed, a time course analyzing changes in salivary glands from patients is required to understand how these organs are affected in the short term and long term. However, our results suggest that targeting these stem cells and their innervating nerves to control and sustain tissue regeneration in response to radiation damage may provide a means of maintaining/repairing tissue for the long term.
A number of recent studies have aimed to address gland regeneration after radiation damage in the mouse model by either isolating putative stem cell populations for reimplantation (Nanduri et al, 2013, 2014) or sparing the regions within the gland that are thought to harbor the stem cells (van Luijk et al, 2015). However, the identity of these endogenous stem cells and whether they contribute to the acinar cell compartment was unclear. Furthermore, the effect of such manipulations on salivary gland innervation has not been reported. Based on our study, it is possible that unintentional increases in innervation due to tissue perturbation lead to maintenance and expansion of SOX2+ progenitor cells that regenerate acini to restore salivary function. Xiao et al (2014) reported recovery of murine salivary function and architecture after radiation‐induced damage with the addition of glial‐derived nerve factor (GDNF), a neuroattractive factor (Knox et al, 2013). However, a recent study demonstrated that GDNF itself does not protect SG stem cells from radiation‐induced damage directly (Peng et al, 2017), suggesting that such outcomes could be the result of improvements to the supporting niche.
Together, our study highlights the extensive regenerative capacity of salivary glands that occurs through the expansion and differentiation of a progenitor cell population, even in the face of genotoxic shock. Based on these data, we propose that by directly targeting SOX2+ cells within the tissue, or by isolating and expanding these cells for transplantation and activation, we might regenerate the secretory units of salivary glands and return quality of life to the patient. This would also require maintenance of parasympathetic nerves, as proposed previously (Knox et al, 2013), to sustain the SOX2+ population. Given organs, such as the intestine, glandular stomach, trachea, and taste buds express SOX2, are heavily innervated by the autonomic nervous system and are damaged by therapeutic radiation for the elimination of cancers, such a strategy may be applicable to the repair of multiple organ systems.
Materials and Methods
Mouse lines
All procedures were approved by the UCSF Institutional Animal Care and Use Committee (IACUC) and were adherent to the NIH Guide for the Care and Use of Laboratory Animals. Mouse alleles used in this study were provided by The Jackson Laboratory and include Sox2eGFP (Arnold et al, 2011), Sox2CreERT2 (Smith et al, 2009), Sox2fl/fl (Taranova et al, 2006), Rosa26mTmG (Muzumdar et al, 2007), Rosa26DTA (Wu et al, 2006), and KitCreERT2 (Klein et al, 2013).
Animal experiments
Adult female mice (aged between 6 and 8 weeks) were used in all experiments, unless otherwise stated. Mice were housed in the University of California San Francisco Parnassus campus Laboratory Animal Resource Center (LARC), which is AAALAC accredited. Mice were housed in groups of up to five per cage where possible, in individually ventilated cages (IVCs), with fresh water, regular cleaning, and environmental enrichment. Appropriate sample size was calculated using power calculations. For transgenic studies, sample size was restricted by length of time required to breed enough animals of the required genotype and gender. Wild‐type animals were randomized into experimental groups using Microsoft Excel software. Transgenic animals were assigned to groups based on genotype. All animals were given a unique ID number and as such were blinded to the researcher during analysis.
Genetic ablation of Sox2 or SOX2+ cells.
Conditional ablation of Sox2 was achieved by injecting Sox2CreERT2;Sox2fl/fl:R26mTmG mice with 2.5 mg/20 g tamoxifen each day for four consecutive days, and every other third day, before euthanizing on day 18. Ablation of SOX2+ cells was performed by injecting Sox2CreERT2; Rosa26DTA:R26mTmG mice with 2.5 mg/20 g tamoxifen each day for four consecutive days, before euthanizing on day 7. The Rosa26mTmG mouse provides a valuable tool to lineage trace in conjunction with cell ablation/recombination (Muzumdar et al, 2007). In brief, this model consists of a double‐fluorescent Cre reporter that expresses membrane‐targeted tandem dimer Tomato (mT) prior to Cre‐mediated excision and membrane‐targeted green fluorescent protein (mG) after excision (Fig EV1D). Thus, in these mice, cells where recombination has occurred and are thus lacking Sox2 or expressing DTA will express GFP. Endogenous GFP was imaged in experiments where cryosections were used, while a GFP antibody was used for paraffin‐embedded tissue (chicken anti‐GFP; 1:500, Aves Labs, GFP‐1020).
Lineage tracing of SOX2+ cells.
Sox2CreERT2;Rosa26mTmG mice were injected with 2.5 mg tamoxifen and euthanized after 24 h, 14 days, or 30 days.
Lineage tracing of KIT+ cells.
KitCreERT2;Rosa26mTmG mice were injected with 2.5 mg tamoxifen daily for four consecutive days and euthanized after 14 days or 6 months.
In vivo denervation experiments.
C57BL/6 or Sox2eGFP mice were administered with analgesics 30 min prior to surgery (carprofen and buprenorphine; Patterson Veterinary and Buprenex; 0.1 and 100 mg/kg (IP), respectively) and anesthetized via inhalation with 2% isoflurane/O2 mix. The surgical area was shaved and prepared for incision with alternating iodine and alcohol washes, followed by local anesthetic (lidocaine; Hospira Inc., 8 mg/kg). An incision was made anterior to the ear and the chorda tympani located as previously described (Klimaschewski et al, 1996) and completely transected using spring scissors. The skin was sutured using non‐absorbable silk sutures (Ethicon) and the wound further covered by surgical adhesive (Vetbond). The contralateral nerve was left intact as a control. Mice were euthanized after 7 or 30 days.
Pilocarpine experiments
Adult male C57BL/6 mice (aged between 6 and 8 weeks) were treated with pilocarpine (Sigma‐Aldrich, P0472; 0.68 mg/ml in 0.9% sterile saline). Mice were anesthetized using isoflurane and injected I.P. with pilocarpine at a dose of 4.5 mg/kg bodyweight (200 μl), or 0.9% saline as a vehicle control. Mice were euthanized after 18 h and glands processed for immunofluorescent analysis.
Gamma‐radiation experiments
C57BL/6 mice were anesthetized with 1.25% 2,2,2‐tribromoethanol (Alfa Aesar) in 0.9% saline (Vedco Inc.). The mice were irradiated using a 137Cs source by being placed into a Shepherd Mark‐I‐68A 137Cs Irradiator (JL Shepherd & Associates). Two lead blocks, positioned 1.5 cm apart, were used to shield the body and the very anterior part of the mouth (the snout) of the mice and expose only the neck and part of the head. The 1.5 cm opening was centered at position 3 (20 cm from the 137Cs source, 15.5 cm from the width of the irradiator cavity). Mice were exposed to two doses of 5 Gy at a dose rate of 167 Rads/min for 2.59 min (one of each side of the head, bilateral, and sequential but on the same day) for a total dose of 10 Gy, to irradiate the salivary glands. This dose was calculated by isodose plot mapping (dose distribution), provided by the manufacturer, and EBT films (Brady et al, 2009) were used to localize the 100% region of exposure for mouse placement. Control mice were anesthetized as per experimental mice, but did not undergo radiation treatment. All mice were allowed to completely recover before returning to normal housing and were given soft diet ad libitum (ClearH2O). Mice were euthanized after 1 h or 1, 3, 7, 14, or 30 days.
Organ culture experiments
Ex vivo linage tracing in adult salivary gland.
Salivary glands from Sox2CreERT2;Rosa26mTmG mice (recombination induced 24 h prior) were mechanically dissected into < 1‐mm pieces, placed in complete media in the presence or absence of 200 nM CCh (Sigma‐Aldrich) or 10 μM 4‐DAMP (Tocris), and cultured for 48 h before being fixed for immunofluorescence. In some cases, mice were irradiated with three doses of 5 Gy before glands were taken for culture as above.
Human salivary gland tissue isolation and culture.
Adult human salivary gland was obtained from discarded, non‐identifiable tissue with consent from patients undergoing neck resection (age 28–78, male and female). Informed consent was given by all subjects and experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Patients had either had no irradiation therapy (non‐IR) or fractionated radiation therapy (IR) < 2 years prior to surgery. Tissue was immediately placed in 4% PFA, RNAlater (Qiagen) or DMEM (Thermo Fisher) for live cell explant culture. For ex vivo culture, non‐IR tissue was dissected into < 1‐mm pieces and cultured in serum‐free DMEM/F12 containing holotransferrin and ascorbic acid. For explant assays, the tissue (SMG and PG) was incubated with 50–200 nM CCh (200 nM CCh shown in results) for 4 h before being lysed for RNA. For salivary gland explant‐parasympathetic submandibular ganglia (SMG) co‐culture, tissue was dissected into < 1‐mm pieces and cultured on a floating filter above serum‐free media. E13 mouse parasympathetic submandibular ganglia were isolated as previously described (Knox et al, 2010). One parasympathetic submandibular ganglion per explant was placed next to the human salivary gland and cultured for 7 days and either fixed for immunofluorescent analysis or lysed for RNA.
Tissue processing.
After fixation, salivary glands (human and mouse) were either processed for OCT or paraffin embedding. For generation of frozen sections, tissue was incubated in increasing concentrations of sucrose (25–75%) and embedded in OCT. 12‐μm sections were cut using a cryostat (Leica) and stored at −20°C. Tissue for paraffin was dehydrated by incubating in increasing concentrations of ethanol and subsequently Histo‐Clear (National Diagnostics) before embedding in paraffin wax (Sigma‐Aldrich). 12‐μm sections were cut using a microtome (Leica) and stored at room temperature.
Immunofluorescent analysis.
Whole‐mount salivary gland and tissue section immunofluorescence analysis has been previously described (Knox et al, 2010). In brief, tissue was fixed with either ice‐cold acetone/methanol (1:1) for 1 min or 4% PFA for 20–30 min followed by permeabilizing with 0.1–0.3% Triton X. Tissue was blocked overnight at 4°C with 10% Donkey Serum (Jackson Laboratories, ME), 1% BSA (Sigma‐Aldrich), and MOM IgG‐blocking reagent (Vector Laboratories, CA) in 0.01% PBS–Tween 20. Salivary glands were incubated with primary antibodies overnight at 4°C: goat anti‐SOX2 (1:200, Neuromics, GT15098); goat anti‐SOX10 (1:500, Santa Cruz Biotechnology, sc‐17342); mouse anti‐TUBB3 (clone TUJ1 at 1:400, R&D Systems, MAB1195); goat anti‐GFRα2 (1:100, R&D Systems, AF429); rabbit anti‐tyrosine hydroxylase (1:100, Millipore, AB152); rat anti‐E‐cadherin (1:300, Life Technologies, 13‐1900); rabbit anti‐EGFR (1:200, Abcam, ab52894); rabbit anti‐KRT5 (1:1,000, Covance, PRB‐160P); rat anti‐KRT8 (1:200, DSHB, troma I); mouse anti‐KRT7 (1:50, Covance, MMS‐148S); rat anti‐CD44 (1:200, BioLegend, 103001); mouse anti‐Ki67 (1:50, BD Biosciences, 550609); rabbit anti‐CCND1 (1:200, Abcam, ab16663); rabbit anti‐caspase‐3 (1:100, Invitrogen, 34‐1700); rabbit anti‐AQP3 (1:400, Lifespan Biosciences Inc., LS‐B8185); rabbit anti‐AQP5 (1:100, Millipore, AB3559); goat anti‐MUC19 (1:200, Abcore, AC21‐2396); mouse anti‐αSMA (1:400, Sigma‐Aldrich, C6198); chicken anti‐GFP (1:500, Aves Labs, GFP‐1020); rabbit anti‐CHRM3 (1:1,000, Research and Diagnostics, AS‐3741S); and rabbit anti‐MIST1 (1:500, gift from Stephen Konieczny, Purdue University). Antibodies were detected using Cy2‐, Cy3‐, or Cy5‐conjugated secondary Fab fragment antibodies (Jackson Laboratories) and nuclei stained using Hoechst 33342 (1:1,000, Sigma‐Aldrich). Fluorescence was analyzed using a Leica Sp5 confocal microscope and NIH ImageJ software.
Morphometric analysis and cell counts.
For immunofluorescent analyses (e.g., Fig 2D), cells positively stained for markers were counted using ImageJ. Acinar cell size was measured using ImageJ. All data were obtained using 3–5 fields of view/group, and each experiment was repeated three times. For nerve density analysis, immunofluorescence of nerve marker TUBB3 was analyzed and raw integrated density (displayed as arbitrary units, AU) was calculated using ImageJ.
Quantitative PCR analysis.
RNA was isolated from whole tissue using RNAqueous Micro Kit (Ambion). Total RNA samples were DNase‐treated (Ambion) prior to cDNA synthesis using SuperScript reagents (Invitrogen, CA). SYBR green qPCR was performed using 5 ng (mouse) or 4–10 ng (human) of cDNA and primers designed using Primer3 and Beacon Designer software or described on PrimerBank (http://pga.mgh.harvard.edu/primerbank/). Primer sequences are listed in Appendix Tables S1 and S2. Melt curves and primer efficiency were determined as previously described (Hoffman et al, 2002). Gene expression was normalized to the housekeeping genes S18 and S29 (Rps18 and Rsp29) or Gapdh for mouse and GAPDH for human and to the corresponding experimental control. Reactions were run in triplicate and experiments performed 2–3 times.
Flow cytometry.
Adult mouse sublingual salivary glands (CD1) were dissected and washed in PBS containing gentamicin (Sigma‐Aldrich). Cell isolation and flow cytometry were performed as previously described (Muench et al, 2002; Pringle et al, 2011). Briefly, a single cell suspension was created by mincing tissue in HBSS + 1% BSA (Sigma‐Aldrich) with a scalpel blade and incubating in a HBSS solution containing 50 mM CaCl2 (Sigma‐Aldrich), 40 mg/ml hyaluronidase (Sigma‐Aldrich) and 23 mg/ml collagenase type II (Sigma‐Aldrich) at 37°C for 15–45 min. The enzyme reaction was quenched by the addition of BSA and the solution filtered through a 70 μm strainer (BD Falcon) and centrifuged at 400 g for 8 min. The resulting cell pellet was washed with sterile HBSS + 1% BSA, centrifuged and resuspended in blocking buffer (5% serum and 0.01% NaN3, BioLegend). Cell surface staining was achieved by incubating cell suspensions with antibodies against CD326 (EpCAM; Miltenyi, 130‐098‐113), rabbit anti‐CHRM1 (Research and Diagnostics, AS‐3701S), rabbit anti‐CHRM3 (Research and Diagnostics, AS‐3741S) and rabbit anti‐AQP5 (Millipore, AB3559). Subsequently, intracellular staining was achieved following fixation and permeabilization using an intracellular staining kit (eBioscience, 00‐5523‐00) and antibodies against SOX2 (BD Pharmingen, 562195) and Ki67 (BioLegend, 652405). Flow cytometry was performed on a LSRII (BD) using the appropriate single stained controls and data collected using FACSDiva (BD) and analyzed using FlowJo. 100,000 events were collected for each sample unless otherwise stated.
Statistical tests
Normal distribution was assessed using the D'Agostino‐Pearson omnibus test. Data were analyzed for statistical significance using Student's t‐test (unpaired, two groups) or one‐way ANOVA (multiple groups) with post hoc testing performed using Dunnett or Tukey tests (GraphPad Prism or SPSS). For multiple testing, we used a false discovery rate of 0.05. All graphs show the mean + standard deviation (SD) or mean + standard error of the mean (SEM), as indicated in the legends.
The paper explained
Salivary gland acinar cells are inadvertently destroyed by radiation treatment for head and neck cancer, resulting in a lifetime of hyposalivation and co‐morbidities. A potential regenerative strategy for replacing injured tissue is the reactivation of endogenous stem cells by targeted therapeutics. However, the identity of these cells in the salivary gland, whether they are capable of regenerating the tissue, and the mechanisms by which they are regulated are unknown.
In this study, we show that SOX2+ cells are essential stem cells for the saliva‐synthesizing acinar cells in the murine sublingual gland and that they repopulate acini following radiation injury. SOX2‐mediated acinar cell replacement is dependent on parasympathetic nerves and that application of a muscarinic mimetic in the absence of nerves is sufficient to drive regeneration. Although SOX2 is limited to the murine sublingual gland, we show that SOX2+ cells are present in all three major human salivary glands (submandibular, parotid, sublingual) and that expression of SOX2, along with parasympathetic nerves, is diminished in irradiated biopsies. Crucially, the addition of a muscarinic mimetic increases the expression of SOX2 and markers of gland function in human salivary gland biopsies. Together, these results suggest that salivary tissue degeneration in human radiotherapy patients is due to loss of progenitors and cholinergic signaling.
This study addresses an area of significant unmet clinical need. Based on these data, we propose that by directly targeting SOX2+ cells within the tissue or by isolating and expanding these cells for transplantation and activation, we might regenerate the secretory units of salivary glands and return quality of life to the patients following irradiation therapy. Given organs, such as the intestine, glandular stomach, trachea, esophagus, and taste buds express SOX2, are heavily innervated by the autonomic nervous system and are damaged by therapeutic radiation for the elimination of cancers, such a strategy may be applicable to the repair of multiple organ systems.
Author contributions
EE, AJMay, and SMK designed and planned the study; EE, AJMay, LB, NC‐P, SN, and AJMattingly performed the experiments; EE, AJMay, LB, and SMK analyzed and interpreted the data; JLC, WRR, and ADT provided tissue samples; EE and SMK prepared the manuscript, with input from AJMay.
Conflict of interest
The authors declare that they have no conflict of interest.
Expanded View
Expanded View Figures PDF [emmm201708051-sup-0002-EVFigs.pdf]
The authors would like to acknowledge Drs. Rushika Perera, Licia Selleri, Rolf Zeller, Andrei Goga, and Scott Oaks for their critical reading of the manuscript. We also acknowledge Alvin Wong for his advice. Funding was provided by a CIRM postdoctoral fellowship (EE) and NIDCR 5R21DE025736 (SMK) and 5R01DE024188 (SMK).
FundingHHS|NIH|National Institute of Dental and Craniofacial Research (NIDCR)http://dx.doi.org/10.13039/100000072 5R21DE0257365R01DE024188
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- © 2018 The Authors. Published under the terms of the CC BY 4.0 license


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Roads across the metropolitan area have becomes slicked and snow-packed as snow from the second major winter storm moves across the Kansas City area.
"Delay, delay, delay the day and push everything back," said Steve Porter, senior customer relations specialist with the Missouri Department of Transportation.
If you don't have to get out, Porter suggests staying home. Snow is expected to continue to fall through the rush hour.
"We have been staying right up with the snow — we are not ahead of it by any means," Porter said before 6 a.m. "There's about 7 inches of snow in Cass County, where there was the heaviest snowfall.
"About 5 inches of snow north of the river at KCI airport. We have been pushing it all night," he said.
Unlike the first storm where people abandoned their cars, road crews have not been impeded by a high number of stranded or stalled vehicles.
"Most of the people were off the roads overnight, which made it much better for crews to push the snow through the night," Porter said.
But the high wind, which was not a factor in the previous storm, is expected to cause drifting snow.
"We suggest people stay off the highway and out of the way while we do the work," he said. "Crews will continue to plow after snowfall ends. We will have a lot of drifting and a lot of pushing the snow off the main lines."
The priorities are to make sure the major highways and interstates are passable, then crews will focus more on the shoulders and minor routes.
"We hope to be on top of that by mid- to late-afternoon — maybe sooner if the we get cooperation from the wind," Porter said.
In Kansas, the roads are snow-packed and are going to be that way for a while, said Kimberly Qualls, Northeast Kansas public affairs manager for the Kansas Department of Transportation.
"Crews are plowing, plowing and plowing," she said. "We can't start treating the roads (with chemicals) until the plowing is done."
Snowplows have been driving side-by-side, but when they go back, the roads are covered back up because of the drifting snow.
"We encourage drivers to stay home," she said. "That is the best thing to do today. The storm will move out at some time, but until then it's not pretty out there at all."
Road conditions in the Kansas City Metropolitan area and Douglas County are completely snow packed with ice. Topeka is reporting wet and slushy conditions.
"With the wind, there can be low visibility to white-out conditions," she said. "There's lots and lots of snow."
The bulk of the snow is expected to end around 9 a.m., but snow showers are expected later in the day and into Wednesday, Porter said.
Kansas City police dispatchers said they were on the scene of a non-injury crash at North Chouteau Trafficway and North Belmont Road before 6 a.m.
Meanwhile, Overland Park police said they had reports of a couple of crashes, but none were serious.
There are reports of the occasional stalled vehicles and tractor-trailers, which are more on the exit ramps.
"The big snowstorm is expected to push out of here before noon," Porter said. "But the horizontal snow caused by high winds will continue until sunset."


text:
Dreams I Have for My Daughter
My husband and I love "MasterChef."
There. I've said it.
We love it because it's ridiculous. And just when we think it can't get any more ridiculous, it does. Like that time Joe Bastianich threw a pile of money into a frying pan, drizzled it with oil, and set it on fire in order to illustrate the point that the cash prize at the end of the show was nowhere near as important as the glory of being able to call yourself a Master Chef. I mean really. I can't even.
RELATED: Will She Be Braver Than I?
Every once in awhile, after I finish making dinner, my husband Michael asks me, "Would Gordon Ramsay approve?"
No. No he would not.
Then "MasterChef Junior" happened. And while I questioned the wisdom of allowing one's child to participate in a high-pressure, nationally televised reality competition show, I couldn't turn away.
I mean really.
Now that the second season is up and running, Michael likes to say to our daughter, "Emily—Are you going to be a Master Chef one day?" And maybe she will. Who's to say she won't? After all, the possibility exists that she will surpass us in everything.
I feel that, as parents, we dream bigger for our children than we ever do for ourselves. We try to nudge our children toward certain interests, perhaps ones we also have or ones we wish we were better at. In reality, the probability that our child will share our interests is just as high as the probability that they will love everything that we hate. But there's nothing wrong with dreaming.
Here are just a few of the ludicrous fantasies we hold for our 4-month-old daughter:
1. America's Favorite Dancer
I can't dance worth a damn, and survived merely one season when my mom placed me in ballet lessons as a child. But I sometimes watch "So You Think You Can Dance with Em," dancing her around the room in loops and twirls and think: Maybe you'll be good at it.
2. Rock Star Yogi
I am a vinyasa yoga instructor, and I love it—both the practice of yoga and the teaching of it—to bits. When I was pregnant, I registered for a baby yoga mat and daydreamed of practicing side by side with my daughter in our sun-lit living room. We now go to mommy and me classes together. But will she drop it all like a hot potato as soon as she's old enough to think for herself?
3. Rock Star, Period
When she screams especially loudly, we predict that she will someday be a singer.
My husband and I are both musical. I played piano and clarinet and also sang weddings and funerals way into adulthood. My husband was the lead singer and guitarist of a rock band. Now the only gigs Michael plays are in our kitchen, as he serenades our daughter. When she screams especially loudly, we predict that, because of those strong pipes, she will someday be a singer.
4. Word Nerd
I've loved reading from a young age and am now make my living as a freelance writer and editor. What I really hope for is that Em will love reading as much as I do. But when I handed her a board book version of "Jane Eyre," she slammed it closed with an indignant growl. Are we doomed?
5. Scientist
My husband is into science, and likes to watch "The Universe" with Em. Sometimes, while transporting her between the car and the house, he'll point up at the sky and show her the stars. He already owns several of the "Geek Dad" books.
6. Athlete
This has nothing to do with me or my husband and everything to do with our families, who think it would be hilarious if the two most unathletic, sports-disinterested people ever ended up with a kid who loves sports. My family is obsessed with baseball. My father-in-law and sister-in-law run marathons. My brother wants to take Em paintballing. There is a pair of Yankees socks in Em's dresser drawer. Ugh.
RELATED: The '80s Movies I Can't Wait to Show My Daughter
7. Web Developer
We both work online, my husband as a web developer and me as a writer. Em certainly gets plenty of inadvertent screen time, which I feel endlessly guilty about. Will she pick up the habit and end up in a pretty lucrative industry, like her dad? Or will she rebel completely and go full-on Luddite?
Only time will tell.


text:
Saturday, 16 February 2019
Last updated 43 weeks ago
Jun 29 2011 | 1:56pm ET
UCM Partners, a boutique fixed-income investment advisor with about $1.1 billion AUM, has launched the UCM MIDAS Fund with $10 million.
The new fund, which targets returns of 25%-30%, is a private, co-mingled fixed-income vehicle designed to capitalize on relative value opportunities in residential mortgage-backed securities and other securities debt markets.
The MIDAS fund is managed by UCM Partners CIO Jay Menozzi and portfolio managers Boris Peresechensky and Vesta Marks. The team employs an investment process based on UCM's proprietary analytical models, developed to capitalize on undervalued cash flows of securities at the top of the capital structure within the residential MBS market.
A company statement says the investment team employs a quantitative relative value approach, utilizing their proprietary loan-level credit models to generate security-level cash flow projections for multiple economic scenarios—expected-base case, extreme-stress case and optimistic recovery case. The fund also seeks to exploit periods of market illiquidity and pricing anomalies to capture opportunities within distressed sectors and among mispriced securities.
Founded in 1992 and headquartered in New York, UCM Partners also has offices in Orlando and Philadelphia.
Jan 30 2018 | 9:49pm ET
As the U.S. shifts from monetary stimulus to fiscal stimulus, market pricing should...
May 24 2017 | 9:25pm ET
Starting in 2019, financial industry executives sitting for the coveted Chartered...
Feb 14 2018 | 9:57pm ET
Tasked with delivering returns on client capital, a common dilemma for many alternative...


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Memorial Houston Homes For Sale
About Memorial, Houston Texas
The Memorial area is one of the most prestigious areas of West Houston. Bordered to the north by I-10 to the south by scenic Buffalo Bayou to the east by 610 and to the West by Highway 6 the community is positioned directly in the heart of Houston's projected growth. A drive through the Memorial area will enchant guests with mature trees, palatial homes and gorgeous bayou vistas. Memorial is the home to not only home to some of the Houston sought after communities, it is also home to job growth, some of Houston's best amenities and some of Texas most recognized schools.
The History of Memorial Area Real Estate
First, to understand the area, you must take a brief walk down memory lane. Many wealthy area business men built homes on large lots and with horse stables in the 1930's. Longtime residents still reminisce about taking horseback rides along Buffalo Bayou. Although the stables are almost all gone and many of the lots have been parceled to allow room for even more gorgeous homes, the community has maintained its stateliness and beauty.
Memorial Area Neighborhoods
Memorial is the home to several charming independent villages like Spring Valley, Piney Point, Bunker Hill, Hedwig, Hilshire and Hunters Creek. The villages boast high end homes to multi-million dollar estates. Additionally, phenomenal subdivisions can be found throughout the area, many with easy access to the gorgeous Terry Hershey Park. Some of these communities include Nottingham Forrest, Thornwood, Memorial Bend, Memorial Drive Acres, Memorial Way, Westchester, Whispering Oaks, Winding Brook, Woods on Memorial, Yorkshire, and Fleetwood. Many home owners can find a wonderful home within Memorials confines. Prices range greatly. From smaller townhomes to estates on acreage the community truly offers homes in most price ranges.
Real Estate In The Memorial Area and The Energy Corridor
Next, the area is defined by job growth. The world famous Houston Energy Corridor resides within the confines of Memorial and is home to some of the world's largest energy companies including Conoco Phillips, BP America, Shell and many more. The West Houston Medical Center has also recently blossomed and is the home to branches of Methodist l, Memorial Herman, and Texas Children's Hospital.
Amenities in The Memorial Area
Additionally the area is full of fantastic amenities. Job growth in the area has led to recent development like the Houston City Center, which is a collection of high end dining, luxury shopping and entertainment venues and city style living. Nearby Memorial City Mall, long considered one of the premier malls in Houston is also a large part of the areas success. Still some of the best amenities come naturally. Buffalo Bayou which wistfully cuts though the area defines the areas beauty. Many of the areas parks and walking trails offer nearby homeowner's wonderful opportunities to hike, bike or simply relax under arbors of the areas native trees.
Memorial Area Schools
Finally, few areas succeed in the long term without great schools. Much of the area is zoned Spring Branch ISD and many of the rest are zoned to Katy ISD. These two school districts have long been two of the best in the state. Additionally, these homes are zoned to some of the school districts best High Schools like Memorial, Stratford and Katy Taylor. The elementary and middle schools are also very strong.
In conclusion Memorial is and will continue to be a premier Houston address. However, like many areas real estate values very greatly. A good Realtor® is needed to traverse the area and insure your investment… A bad decision in the area could cost a homeowner thousands of dollars but a great decision can lead to a real estate Nirvana. Contact our team today for a free preview.


text: AirBuds are guaranteed to be the most comfortable earbuds you own. If you are not satisfied with the comfort level of our earbuds please send them back within 30 days for a full, 100% refund. If you are having any issues whatsoever, we encourage you to contact us and our personable customer service representatives will help.


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Today has been a big improvement over yesterday. Immediately after getting up, my left eye still had the somewhat painful "eyelash" sensation, but by mid-morning that had gone away and today is the first day I haven't had to take any pain killers - just steroidal drops (which I will be on for months to come). My vision is getting better day-by-day. Today I could read the address numbers on houses next door!
I went for the last of the daily follow-ups with my doctor and, besides apologizing profusely for the incident yesterday, he said everything is healing up just as expected. The left eye is now a day or so behind the right eye because of tearing the epithelium, but it has healed up and is coming along. I still have a lot of haloing - especially with the left eye, but the improvement is very noticeable now. Amazingly, he said I actually would be legal to drive! Frankly, I still find the idea slightly terrifying and I would give it a few days yet before I will be back to driving.
So, all-in-all, good progress and now moving more quickly! I will likely stop posting about this every day now and just give periodic updates as my recovery continues.
Tuesday, June 1, 2010
PRK - Post surgery Day 4
Posted by Torchris at 5:23 PM
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You can choose from a wide variety of styles or send them a message with your very own custom order.
I was able to review their Mommy-n-Me necklace set in mint and white (so hot right now), and the necklaces are just too cute for words!
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As a workaround, I've now implemented a better detection mechanism based on the sysfs path of the hidraw file. For Bluetooth devices, the path could look like this:
For USB devices, it could look like this:
In the case of the PS Move Motion Controller, the vendor ID is 0x054c and the product ID is 0x03d5. I've highlighted the occurence of these IDs in boldface above. Given that the hidapi already determines the sysfs path of the hidraw device, it's easy to write a function that extracts the IDs from the path. I've done so now in a patch against hidapi: commit 8ba92edb519 in thp/hidapi.
In order to get it merged into hidapi upstream, I've created pull request #62 at the signal11/hidapi repository on Github. Let's see if I have to rework/improve the patch or if it will be accepted.
After the patch has been merged (you can merge it locally or clone from my hidapi repo), connecting to the PS Move on Linux will become even easier. The only part remaining then is figuring out the permission problems on the hidraw devices (we could probably fix this with some udev rules) and getting Bluez' bluetoothd to reliably accept connections from new PS Move controllers.


text: I've been documenting the final days before my wedding on April 25 (less than two weeks now!), and I have a lot to report in this post. Last Friday was my final wedding gown fitting at the Reem Acra salon here in NYC. I won't go into much detail about the dress in case my resourceful fiance finds his way onto this blog—except to say that it is beyond stunning and I couldn't be more excited to wear it. Which probably sounds like the usual gushing, but it's actually a big deal since after I first made the purchase (by far the biggest fashion splurge of my lifetime) almost a year ago, I confess, I had dress doubts. To understand my second-guessing, you have to know that I have chronic indecision. I stress over what to order at restaurants (and have been known to furiously flag down waiters to change my entree). I spent hours in Ikea picking out basic bookshelves and a coffee table for our apartment (black or dark brown? a perfect match or close complement?). Even registering was agony, despite the fact that—as my fiance repeatedly and exasperatedly pointed out???nothing was set in stone. So it's not surprising that The Dress Decision was a difficult one for me. I must have tried on 40-plus gowns all around town to make sure I tested every style imaginable. And I fell in love with a handful. I kept waiting to have that moment of clarity that many brides describe and some of you may have experienced—standing in front of the mirror and knowing this dress was it. Instead I felt like a million bucks in so many I couldn't choose. When I finally forced myself to pull the trigger on the clear front-runner (it perfectly suits the outdoor setting of our wedding), I fretted that I'd been overly influenced by my 'maids and mom rather than following my gut. What it took to put my mind at ease was a stealth solo trip back to the Reem salon (thank you Heidi and Holly for not calling me crazy!) to try the dress on one final time. After that, I never looked back. And nine months and four fittings later, I am 100% sure I made the right choice. Now if only I could decide on a good gift for our ring bearers!—Elaine Stuart, Senior Associate Features/Travel Editor, Modern Bride Magazine


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text: Intellectual Development in a Danish Cohort of Prematurely Born Preschool Children: Specific or General Difficulties?HOFF ESBJØRN, BARBARA Cand. Psych., Ph.D.1; HANSEN, BO MØLHOLM M.D., Ph.D.2; GREISEN, GORM M.D., Dr. Med. Sci.2; MORTENSEN, ERIK LYKKE Cand. Psych.3Journal of Developmental & Behavioral Pediatrics: December 2006 - Volume 27 - Issue 6 - p 477-484 ORIGINAL ARTICLES Abstract Author InformationAuthors A national cohort of extremely low birth weight (ELBW) and/or extremely preterm (EPT) children and a term control group was followed up at the age of 5 years. The primary objective was to investigate whether premature birth had a global impact on cognitive functions or affected specific functions only. Assessment tools were Wechsler Preschool and Primary Scale of Intelligence-Revised (WPPSI-R), Movement Assessment Battery for Children (M-ABC), and subtests from the Neuropsychological Assessment 4-7 years (NEPSY). The mean Full Scale IQ (FSIQ) and M-ABC score of the index children were 1.1 and 1.2 SDs lower than that of the control children (p <.001). Most WPPSI-R subtests showed medium to large differences between index and control children, suggesting a global impact of premature birth on cognitive functions. For both unadjusted and FSIQ adjusted means, no significant group differences on tests of memory or executive function were observed (p >.1), suggesting little impact of premature birth on these specific functions. In this sample, cognitive difficulties in 5-year-old ELBW and/or EPT children tended to be associated with general intellectual difficulties rather than with specific dysfunctions; however, the implications of this finding are ambiguous due to substantial attrition on the NEPSY subtests. 1Department of Psychology, University Clinic, University of Copenhagen, Copenhagen, Denmark 2Department of Neonatology, Rigshospitalet, Copenhagen, Denmark 3Department of Health Psychology, Institute of Public Health, University of Copenhagen, Copenhagen, Denmark Received July 2004; accepted July 2006. Address for reprints: Barbara Hoff Esbjørn, Cand. Psych. Ph.D., Stampevej 31, 2970 Hørsholm, Denmark; e-mail: firstname.lastname@example.com. © 2006 Lippincott Williams & Wilkins, Inc.


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Typically true to size, some room for layering
Insulated - ideal for the cold, layers urged in extreme cold
Lightly treated, okay for light snowfall or rain
Minimal sweat evaporation during activity
With a unique parka fit the Oakley Code Insulated Jacket is fashionable for everyday use, with performance features built for the mountain. Featuring Hydrogauge 10 which offers 10,000mm of waterproofing and 10,000g of breathability while 60/80g of Thinsulate insulation keeps your body warm. Mesh-backed underarm vents and an adjustable hood provides ventilation and protection from the elements, while the interior storm skirt no unwanted snow. The Code Insulated Jacket is equipped with plenty of pockets for specialized storage and critically taped seams that block out water and offer outstanding durability all season long.


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Two Bradley University economics professors on Friday defended their economic impact study of the proposed Peoria Riverfront Museum and Caterpillar visitors center.
Two Bradley University economics professors on Friday defended their economic impact study of the proposed Peoria Riverfront Museum and Caterpillar visitors center.
The study was criticized by fellow economists Thursday.
"After we looked at what they wrote about our study, we don't see anything that would fundamentally change what we see as a wonderful opportunity for the community to build the museum on the Downtown block," said Robert Scott, the chairman of the university's economics department.
Said Joshua Lewer, the co-author of the study:
"We certainly stand by our work."
On Thursday, the museum opposition group Citizens for Responsible Spending released two separate analyses of the Scott-Lewer study, one by Knox College economist Richard Stout, the other by the team of Lon Carlson and Frank Beck of Illinois State University. The critiques were done at the request of members of the museum opposition group, who mailed them the Bradley study. Two professors at the University of Illinois did not respond to the request.
Both studies cast doubt on the claim by the Bradley report that the project would have a positive economic effect of $575 million over 20 years. A story in Friday's editions incorrectly stated that effect. If voters approve a sales tax increase of a quarter of a percent in April that would raise $40 million for the museum and then be retired in 20 years or sooner, the burden on the average resident of Peoria County would be $17 a year. The economic benefit then, according to Scott and Lewer, is a 14-1 return on the taxpayers' investment.
However, the other economists believe a calculation the Bradley professors used to figure the overall economic effect of the project overestimated it. Called a "multiplier," it's the amount of additional spending that will occur in the community for every dollar spent on construction, operating costs and by visitors to the museum block. The spending multiplier used in the Bradley study was 1.70; each dollar of spending causing a total of $1.70 of economic activity.
"Rather large," wrote Carlson and Beck. "The authors need to provide better justification for the numbers selected."
"A spending multiplier of 1.7 is probably optimistic," wrote Stout of Knox College.
OK, Scott and Lewer said Friday, even if the number is high - and they won't concede that it is - go ahead and do the math with a lower figure.
"The two reports both assert that the multiplier we used might be high," Scott and Lewer wrote in a prepared statement. "We think not - it is consistent with other studies and with government data. More important to discuss what the result of a different multiplier assumption would be. Will the ration of benefits to costs drop to 13 to 1? That's still huge."
Lewer and Scott said they were discouraged by the willingness of the economists to publicly criticize their report without benefit of the data and other information they used to write it.
"We think that most of the questions they had would have been answered if they had contacted us and if they looked at the data we used," Scott said. "Our numbers are real numbers. They are not fake statistics."
Scott Hilyard can be reached at 686-3244 or at email@example.com.


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Ines picking up the Scottish game very quickly on pitch 1 of Blood Sweat...Audrey, Matt and Simon on Central Buttress
Saturday, February 20, 2010
Scottish Pride
Ines picking up the Scottish game very quickly on pitch 1 of Blood Sweat...Audrey, Matt and Simon on Central Buttress
6 comments:
- Toby - Northern Light Blog said...
The first picture is pure magic Ian!
- February 21, 2010 at 9:55 AM
- Ian Parnell said...
It was just an extraordinary day. Having persuaded everyone to get up at 4am and leaving the campervans in snow, I was hoping against hope that the forecast would prove right. Then to reach the summit plateau as the sun broke the skyline was one of those occasions where you're beaming away with joy and then a little sob rises up and embarassingly you try to hide it with a cough or a snort! At that point Ines said it was worth the effort just for those moments.
- February 21, 2010 at 10:06 AM
Hi Ian, was wondering who were the lucky folks in the van on thursday night! We didnt pull in till midnight. Was that 2 of the guys getting back at 1am? Think we also saw your lights heading up the hil in the morning.... wondered what mega route you were heading for! Nice one! So is B, S and F tears just 3 hard VII pitches? Well protected?
- February 21, 2010 at 8:53 PM
- Luca l'Alpinauta said...
great pics and greats pitches
- February 21, 2010 at 9:22 PM
- Ian Parnell said...
I think that might have been Roger Webb who did a new thing on Liathach or a female pair on Central Butt. We topped out at about 3.30pm and were back to luxuriate in the vans by dark. Blood Sweat is 3 grade VII pitches although none easy, I think in current conditions the 3rd could be the crux. If you are looking for an VIII that won't risk getting downgraded Brusied Violet is at least a grade harder ;-)
- February 21, 2010 at 9:33 PM
Sorry Ian, I read your account of bruised violet therefore its not top of my list!!!!
- February 22, 2010 at 1:14 PM


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The Apple iPad is found in many meeting rooms being clutched by individuals as their life support systems. The iPad contains their email, calendar, and a range of other important business documents. Yet in a meeting the iPad represents an isolating and personal device. With nsquared presenter digital tables, interactive touch screens and presentation screens can display content from an iPad to be shared with the group for discussion.
Here some examples of how people are using nsquared presenter and iPads:
- A sales person keeps a range of product images on their iPad and shares the relevant images on a digital table with customers as required.
- A sales team collates ideas from their personal devices to a digital table to create a new presentation.
- Students collect research materials on their iPads and then gather around their digital table in the library to create group projects.
- A teacher shares question sheets from their iPad to the digital table for a group of students to discuss and answer.
- A designer shares sketches from their iPad with clients around a touch table.
- A project team share progress reports from their iPads to the digital table in their meeting room in their weekly status meetings.
|Sharing content from iPad to digital tables using nsquared presenter


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A 3/8" soft shackle made from Samson's Amsteel Blue, Dyneema SK-75 fiber.
Has an average break strength of 29,200 lbs and a working load limit (WLL) of 7,300 lbs at a 4:1 safety factor.
A great way to attach to tube or other surface (bumper, sliders, cage, ground anchor, etc) where a traditional hard shackle or choking a strap will not work. Comes in colors orange, silver and dark charcoal.
Comes with a section of loose fit abrasion guard. One end has a slip loop which should be securely tightened around the opposite knot end to create a closed device. Position knot and slip loop in free space away from other recovery gear or your attachment point (see picture). Works best on smooth, rounded surfaces. Do not place around sharp objects. Do not use for dynamic (snatch) recoveries.
Not for lifting use or use during any life saving operations.
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Southeast Overland disclaims all liability and responsibility for any injury or damage that may occur when using a recovery product sold by Southeast Overland. Recovery of a vehicle involves extreme loads and can result in injury and death. Please seek professional instruction in the proper use of recovery gear.


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Hi guys!
I've been here in the beautiful country Ireland for nearly two weeks now and I'm enjoying it!
I'm attending a school called Rockford Manor, which is only for Girls, like most of the schools in Ireland. We are supposed to wear school uniforms, which saves a lot of time in the morning. I have to get up at 7:30 am, compared to Austria this is actually luxury! That's because school starts only at 9:00 am, but we have to be in school a Little earlier because the assembly takes place every morning. There the principle tells us general information about the things that are going on in school, while some teachers are checking the student's uniform, nails, and piercings or necklaces. Assembly is finished by the morning prayer and then the normal classes start. On my fist day I had to choose all my subjects, because there are compulsory subjects (Maths, Irish, Englisch, P.E., Religion, Careers and SPHE) and optional subjects - I have picked Business, Art and Geography. Exames have to be done at Christmas and by the end of the school year so they don't really have to study during the year!
The building is really old with many many corners and it is not well equipped with technology or furniture but I am getting used to it. :D
More info will follow.
Lovely Greetings from the Green Island


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1 Bedroom Flat in Mill Street, Kidlington - OX5
One bedroom apartment in converted former Manor House, with high quality finish throughout. Parking space and use of communal garden. Great access to Oxford Parkway Station.
One bedroom apartment in converted former Manor House, with high quality finish throughout. Parking space and use of communal garden. Great access to Oxford Parkway Station.
- Double bedroom with built in storage
- Open plan living/dining room with double aspect and wooden flooring
- Compact kitchen with granite worktops
- Reserved parking space and use of grounds
- Great for access to Kidlington and the Parkway Station
- Furnishing: Fully Furnished
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- Motorway: M40 - J10, 9.17 miles
- Locality: Town/City
- Train: Oxford Parkway, 1.47 miles
- Council Tax: Cherwell, Band B £1,370.23
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To Learn More About Steve Carpenteri
A patron at the local diner recently mentioned to me that "hunting season" was about to start, October being just a week away. That got me to thinking and a check of the Maine 2012 hunting seasons show that things are pretty much already underway. The bear hunting season opened Aug. 27 and the Expanded Zone archery deer season has been open since Sept. 8. The permit-only moose season opens today in certain zones and continues through Nov. 10.
The regular archery deer season opens Sept. 29, with Youth Deer Day (Oct. 20), Maine Resident-Only Day (Oct. 27) and "Everybody Day" Oct. 29.
The Zone 2 fall archery wild turkey season opens Sept. 29 followed by the Zone 1 archery season (Oct. 6), and then the archery-shotgun season opens in Zone 3 Oct. 13.
The ruffed grouse, pheasant, squirrel and hare seasons open Oct. 1, while the raccoon and skunk (??) season opens Oct. 15.
Waterfowl seasons have been underway since early September for resident geese, and will continue in various splits and zones into January.
Point being, of course, that there is plenty to think about if you are a hunter looking for something to do this fall. Most of these sports (with the possible exception of skunk hunting) can occupy the better part of a day if you are serious about it, or they can keep you busy for several hours in the morning or evening if you are just a casual participant.
The young, intrepid types who try to participate in all of these seasons every day all season (which I often did myself back in the 1960s, '70s and '80s) will wear themselves out making the effort along with several pairs of hunting boots.
It's almost a lucky thing that we don't allow Sunday hunting in Maine, but Sunday fishing is still legal and trapping (which pretty much includes the month of November) is also legal. Factor in such things as scouting, game preparation and gear maintenance and it's a sure bet that you won't be doing anything around the house till it's time to put up the Christmas lights!
I spent many a fall running from the deer woods to the grouse thickets to the beaver ponds to the apple orchards in an effort to participate in all the outdoor sports that are legal in October and November and finally came to realize that while it can be done, it's not nearly as much fun as it sounds. Between the different clothing, equipment, strategies and tactics one must use to succeed at any given sport it's a nightmare to try to keep up.
What confounded me most was the steel shot regulation for waterfowl, which requires hunters to possess only non-toxic shot when hunting ducks or geese. Before that law went into effect I could load my vest with shells and wander the woods at will, chasing woodcock in the alders, grouse in the birches and then black ducks or wood ducks in the swamps. Now I have to have one vest filled with lead shot for upland birds and then keep another loaded with non-toxic shotshells when I'm near the water. It's true that one can use steel shot on upland species but those loads are invariably "high brass" magnums intended for thick-feathered birds like ducks and geese and will shred a relatively fragile grouse or woodcock when centered at close range.
My solution was to hunt waterfowl at dawn and dusk and upland birds during the day, which meant changing vests every time I entered new habitat. Toss in a little bowhunting for deer and then rifle hunting, trapping and even fishing at some point during the day and you can imagine what a tangle of gear and clothes I'd have in my truck.
These days I spend the mornings and evenings on some secluded beaver flowage hoping a duck or goose will fly by, and then focus only on deer, bear or upland game during the remainder of the day. I can do more and certainly have done so, but I enjoy pursuing one thing at a time far more than trying to chase them all at once.
Having said that, I notice that my list of necessary chores seems to have gotten shorter as the greater mix of hunting seasons approaches. All the painting, staining, building and fixing has been done, the firewood is in and the wood stove is clean and ready for the season's first real fire. The ceiling fans have been cleaned and reversed (to draw the warm air in from the wood stove) and the back-up heating system is cleaned, primed and ready.
Slowly but surely I have made sure that all my chores, odd jobs and "to-do list" items have been completed, even though there are several weeks of good fall weather ahead. It's as if I have been slowly taking care of all my late-summer responsibilities because another hunting season is just around the corner. Despite my good intentions and better judgment, I'm likely to try to participate in every season that's open just because I may get only one chance to do so before fall, like summer, comes to its surprisingly rapid end.
I should know better after half a century in the woods but I am sure that I will try to do it all at least one day this fall, perhaps two or three times! One morning the day will start out with perfect weather for ducks, the afternoon will be ideal for grouse hunting, and then the hour before sunset will be perfect for sitting in an apple orchard waiting for deer.
Even after 50 years I can't resist the many attractions of fall in Maine. The choices are many but the hours of daylight become shorter with every passing day. Before long the 2012 hunting season will end, and then all that remains will be the memories that were made.
Get out there and enjoy it while you can because it's a long, long time till fall 2013!
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Two Saturdays ago, a hundred or so protesters gathered on the loamy banks of Prospect Park Lake. The afternoon was exceptionally cold, and the group, bundled in heavy coats and hats, formed a knot around a 51-year-old woman named Mary Beth Artz. Artz, who grew up in Brooklyn, is a former actressher credits include the European tour of Hairbut these days is best known as the informal spokeswoman for the park's embattled Canada geese.
In July 2010, biologists with the United States Department of Agriculture surreptitiously rounded up nearly 400 of the birds, including goslings, and trucked them to a gas chamber at JFK. The city, on whose behalf the USDA acted, framed the culling as a matter of aviation safetyit was a mass of Canada geese, for instance, that was to blame for Captain Sully's ditching on the Hudson. But more geese had arrived at the lake, as they do every winter, and although the city had not definitively said it would destroy the animals, its contract with the USDA is good through June 30. Never again, Artz said. That's what I promised myself. Artz insists that the birds in Prospect Park are resident geese, which spend the entire year in Brooklyn and thus pose little danger to airplanes. She suspects a different motive at work. Geese generate an incredible amount of poop, she said. No, it's not pleasant to look at. That doesn't mean we should kill them.
Artz hadn't wanted to hold her vigil now, but the Prospect Park calendar fills quickly, so she had to settle for March, at least two months before the next goose-icide would most likely occur. (In the early summer, geese molt; during that time, they don't fly.) Despite the timing, she had assembled a large and varied group: senior citizens on motorized wheelchairs; a man with a tattoo of a bird on his neck; a gaggle of children, and one golden retriever, wearing STOP KILLING GEESE T-shirts. The geese themselves bobbled in the water nearby.
But why save these geese? Why save any goose? Geese are beautiful, people said. (This is true.) And endangered. (This is not.) But one thing a goose definitely has on, say, a ratwhose extermination is rarely protested, even though rats are closer to us on the evolutionary ladder and, no kidding, have been found to laugh and hold varying degrees of optimism about their own futuresis the simple matter of size.
You may not be able to distinguish the features of a sparrow when it alights on your feeder, says Sy Montgomery, the author of Birdology. But when you get something as big as a goose, you can get a better sense of the personality. And once you start to see personality, you start seeing other things. Geese are unbelievable parents, and they mate for life, says Chris Santopietro, the owner of Geese Relief, a Connecticut company that uses border collies to shoo the birds. And in this day and age, that's something pretty neat. He added, Sometimes their intelligence is incredible, and sometimes they're incredibly stupid. Hey, just like humans.
After a rousing speech, Artz yielded the stage to State Senator Eric Adams. If we save these birds, Adams trumpeted, we save our children, and we save ourselves. The pain and suffering any parent feels when he or she loses a childthat's not isolated merely to human beings. One of these little ducklingshe caught himselfer, birds.
Hatchlings! someone offered.
Hatchlings, Adams agreed. It's the same experience.
A few minutes later, Adams led the crowd in a chant of Give geese a chance. On the outskirts of the rally, a woman shook her head. Don't forget about those possums, she muttered, and walked off in the direction of the street.