Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half  or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.Wood frogs ( Rana sylvatica), a North American species that ranges from the southern Appalachians to within the Arctic Circle. Whereas frogs indigenous to the upper Midwestern United States and southern Canada tolerate freezing only to temperatures as low as -3 to -6°C, frogs from Interior Alaska readily survived experimental freezing to -16°C [Although freeze tolerance in vertebrates was first reported over 30 years ago  and dozeWe hypothesize that extreme freeze tolerance in this northern phenotype derives in part from an enhanced cryoprotectant system that uses urea, which accumulates in autumn and early winter, before freezing occurs, and glucose, which is quickly mobilized from liver glycogen after freezing begins. Both agents colligatively reduce the amount of ice forming in the body, but each also serves other functions that enable cells and tissues to withstand myriad stresses caused by freezing and thawing .-1 [-1) in Alaskan R. sylvatica kept hydrated under humid conditions [-1 plasma) of an osmolyte(s) whose identity was not determined.Urea accumulation is a universal amphibian response to osmotic challenge, such as dehydration or salt acclimation, which serves to limit the transcutaneous loss of body water . Absent -1 . Howevernditions , raisingR. sylvatica is strongly influenced by the quantity of glucose mobilized during freezing. In Alaskan frogs, glycemia rose to 217 μmol ml-1 during a 48-h bout of experimental freezing to -2.5°C [The capacity for freezing survival in o -2.5°C . Howevero -2.5°C . Furthero -2.5°C , individR. sylvatica. We report that urea accrual in frogs preparing for hibernation is subsidized by regulated proteolysis in muscle, a response strongly influenced by water balance. In addition, we demonstrate that glucose mobilization from hepatic glycogen reserves is responsive to severity of the freezing episode, and that multiple freeze-thaw cycles, while not essential to high glucose mobilization, improve the distribution of cryoprotectant to peripheral tissues.This work provides new information about the cryoprotective systems supporting extreme freeze tolerance in a subarctic population of R. sylvatica by hand or dip net from publicly-owned woodlands in Fairbanks North Star Borough, Alaska, USA , between 30 July and 5 August, 2012, under approved methods and appropriate permits issued by the Alaskan Department of Fish and Game. Protocols for experimentation and euthanasia, which was carried out by double-pithing without the use of anesthesia, were approved by the Institutional Animal Care and Use Committee of Miami University (Research Protocol #812).We collected post-metamorphic 2) that was closed with a perforated lid. Each such cage contained a shelter  that rested on two layers of unbleached paper towel, which we replaced at 1-week intervals. Except as noted elsewhere, we wetted this substratum with 2.5 ml dechlorinated water, which thoroughly dampened the fibers and left a thin film of water on its surface.After their capture, frogs were topically treated with tetracycline HCl, placed in plastic cups containing a moist sponge, and shipped under refrigeration to our laboratory, where we removed the sponge and added fresh tetracycline solution. They were kept at ~18°C until the next day, when we transferred each frog to a translucent, polyethylene container  or held until used in November .Following Costanzo et al. , we promad libitum with small (~13 mm) crickets that had been dusted with a vitamin supplement . On three days each week we added as many as five live crickets  and removed any dead crickets from the cages. Some frogs fed more readily than others, but most refused food after the last week in August and so the feedings were discontinued. A few frogs appeared ill or died and were eliminated from the study; we collected data only from healthy individuals.During winter conditioning, frogs were fed N = 32). We assigned each individual to one of three groups: late summer, winter-conditioned/hydrated (HYD), or winter-conditioned/dehydrated (DHYD). Assignments were made in a manner that standardized the SBM mean and variance among groups, but without regard to gender, which could be determined only upon dissection; sex ratio for the pool was 1.5:1.0 .We investigated the effect of hydration state on various physiological adjustments accompanying physiological preparation for winter. To provide sufficient tissue for the analyses, we used the larger of the frogs available for study; for this pool, standard body mass (SBM), determined by weighing frogs after removing any bladder fluid with a cloacal catheter, was 9.3 ± 0.4 g  were euthanized and sampled on the day after their arrival in our laboratory, whereas HYD frogs (N = 10) and DHYD frogs (N = 14) were first subjected to the full winter-conditioning regimen, as described above. HYD and DHYD groups were treated identically except that the latter was exposed to drier conditions. Initially, the substratum in cages with DHYD frogs received only 1.0 ml water, which dampened the paper and humidified the air, but did not provide a free-water surface. When replacing the substratum at weekly intervals, we reduced this amount to 0.5 ml (weeks 2–4), and added no water during the final week. By contrast, HYD frogs were continuously exposed to moist substratum throughout the regimen. We totaled the number of crickets consumed by each frog in both groups. Following winter conditioning, we euthanized all frogs and sampled their tissues as described in the next section.Late-summer frogs  to isolate the plasma, which was promptly frozen in liquid N2. We quickly excised the intact liver, which was lightly blotted on laboratory tissue and weighed on an electronic balance. We also excised two muscles, gastrocnemius and gracilis, from the right hindlimb.Working inside a refrigerated (4°C) room, we determined the SBM of each frog, euthanized it by double-pithing, measured its snout-ischium length, and dissected it to expose the visceral organs. Blood was drawn into heparinized microcapillary tubes from an incision in the aortic trunk and centrifuged . We removed, weighed, and discarded any coelomic fat body. The carcass was then weighed and thoroughly dried so that its water concentration could be determined and, by extrapolation, mass of the entire dry body could be estimated. We measured the length of the tibiofibula of the right hindlimb with dial calipers, this value being used to normalize the mass of the dried gastrocnemius to body size.Portions of the liver and gracilis were immediately frozen in liquid NN = 50) used to feed frogs during winter conditioning.Dried carcasses  were homogenized in a coffee grinder and then pulverized to fine granules with a mortar and pestle. We estimated organic concentration in these samples by burning a weighed aliquot (~125 mg) at 550°C for ~18 h in a muffle furnace  and then reweighing the ash residue. Nitrogen and caloric concentrations were determined via the combustion method and bomb calorimetry, respectively, which were carried out under contractual agreement with the University of Arkansas Poultry Science Central Analytical Laboratory . Total amounts of organic matter, nitrogen, and energy in carcasses were found by multiplying the sample concentrations by the dry body mass. Using the aforementioned procedures, we determined quantities of water, organic matter, nitrogen, and energy in a homogenized sample of the vitamin-fortified crickets  in a 0.2 M sodium acetate buffer  and, after 2 h, the reaction was stopped by adding cold 7% (w/v) perchloric acid. Glucose was determined by glucose oxidase assay and the quantity generated during enzymatic digestion  equated to glycogen concentration, expressed as glucosyl units (μmol g-1 dry tissue). We computed hepatic glycogen content (μmol) as the product of the tissue concentration and dry organ mass, and glycogen richness (μmol g-1 dry tissue) by dividing hepatic glycogen content by dry body mass.We prepared deproteinized extracts of liver and muscle (gracilis) by homogenizing frozen samples in cold 7% (w/v) perchloric acid and then neutralizing the aqueous portion of the homogenate with potassium hydroxide. Extracts were assayed for urea, glucose, and lactate using urease, glucose oxidase, and lactate oxidase procedures , respectively; metabolite concentrations were expressed as μmol gWe analyzed the plasma for the three metabolites mentioned above, as well as total protein, which was measured using the Bradford method  with bovine serum albumin as the standard. Plasma osmolality was measured by freezing point-depression osmometry  using appropriate sodium chloride standards.Analysis of the free amino acid pool in late-summer, HYD, and DHYD frogs was made on samples prepared by combining similar quantities (55–70 μl) of plasma taken, as available, from all or most individuals within each group. The resultant plasma pools were thoroughly mixed, aliquoted into small portions, and frozen (–80°C) prior to further analysis. Amino acid quantitation was carried out using ion-exchange chromatography under contractual agreement with Biochemical Genetics Laboratory, Children’s Hospital Colorado .N = 33; sex ratio, 1.2:1.0 male:female) to experimental freezing using a protocol that ensured frogs froze slowly, a condition that facilitates cryoprotective responses and improves freezing survival, and is ecologically relevant [3), which were covered and loaded into a programmable incubator  set at 0°C. Temperature was continuously recorded using several microprocessor-based loggers  placed within a sham container (lacking a frog) inside each box and on shelves inside the incubator.In mid-November, we investigated certain physiological responses of hibernating frogs  at a uniform rate (0.05°C h-1) to -8°C, a process requiring 160 h, and held them at that temperature for 6 h before dissecting them. In another, we cooled frogs (N = 7) at the same rate to -16°C, a process requiring 320 h, and held them at that temperature for 14 h before dissecting them. We subjected additional frogs (N = 12) to multiple freeze/thaw cycles. Parameters of this experiment were similar to those of the -8°C freezing trial, except that we interrupted cooling when the frogs had reached -4°C and raised the incubator temperature to 4°C. After the boxes had remained at 4°C for 12 h, during which time the frogs had visibly thawed but the ice surrounding them had not completely melted, we reset the incubator to initiate another cooling bout. After three such cycles, frogs were cooled to and held at -8°C for 5 h, following which some individuals (N = 7) were immediately euthanized and dissected; others (N = 5) were thawed at 4°C and monitored for signs of recovery, including exhibition of the “righting reflex” within 2 s of being placed on the dorsum. This multiple freeze/thaw trial lasted 19 d, with frogs experiencing a cumulative 15 d of freezing, assuming they were frozen whilst temperature was ≤ –0.7°C, the approximate freezing point of their body fluids.In one trial, we cooled frogs (N = 7) taken directly from their cages at 4°C, were quickly euthanized, dissected, and processed as described in Morphometrics and Physiological Assays (above), except that blood could not be collected from frozen frogs and no analyses were made on dried carcasses.Frozen frogs destined for physiological analyses, along with a set of reference frogs , followed by Student-Newman-Keul’s Multiple Comparisons Test, to compare data from three groups; data were first log transformed, as necessary, to meet assumptions of normality and homoscedasticity. In the few cases where transformation was ineffectual we used the nonparametric Kruskal-Wallis H-test, followed by a Dunn’s test, to compare values among groups. Significance was judged at P<0.05.Summary statistics are presented as mean ± 1 SEM. We compared data from two groups using a Student’s U = 69.5, P>0.99) in the numbers of crickets consumed by HYD frogs (5.4 ± 1.1) and DHYD frogs (6.0 ± 1.4). On average, each cricket weighed 151.6 mg (fresh mass) and contained 114.3 mg water, 4.21 mg nitrogen, and 0.837 kJ energy.Late-summer frogs, which were dissected promptly after their arrival in our laboratory, had fed recently before their capture, as their guts contained arthropods in various stages of digestion. Frogs subjected to winter conditioning each consumed up to 19 crickets, with much of the feeding occurring earlier in the regimen, when ambient temperatures were higher. There was no difference  in DHYD frogs (15.3 ± 1.8%) than in HYD frogs (6.7 ± 1.6%). Accordingly, water concentrations in tissues of DHYD frogs were consistently lower than those in HYD frogs, although the differences were modest, being only 9% for liver, 16–19% for skeletal muscles, and 14% for carcass  accounted for only half the increment (149 units) in total osmolality. Therefore, HYD frogs apparently co-accumulated with urea a comparable amount of some unidentified solute(s). Plasma protein concentrations in late-summer frogs and HYD frogs were indistinguishable.We examined winter-conditioning effects on plasma levels of select metabolites by comparing values between late-summer frogs and HYD frogs . There wand urea . The sub-1, was inordinately high, being ~88 units above the level resulting solely from dehydration . Thus, during winter conditioning DHYD frogs accrued substantially more urea than HYD frogs primarily by increasing ureagenesis. Plasma osmolality of DHYD frogs, 526 ± 17 mOsmol kg-1, was 152 units higher than that of HYD frogs  among groups, attesting that tissues dehydrated with freezing; water loss was up to 66% in liver, 41–54% in gastrocnemius, and 33–48% in gracilis  than that of unfrozen (control) frogs, 16.8 ± 1.7%. HSI values of frogs in the -8 and -16°C groups  were indistinguishable, but greater than those of the cyclic-freeze group, 6.7 ± 0.3%. Frogs frozen to -8 or -16°C had hepatic glycogen levels only ~32% of those found in unfrozen frogs, 2978 ± 105 μmol g-1 dry tissue  are useful for establishing baseline metabolite levels in the winter-conditioned frogs used in this experiment. Plasma levels of glucose (8.8 ± 2.0 μmol ml-1), lactate (1.1 ± 0.2 μmol ml-1), and urea (74.4 ± 6.4 μmol ml-1), and plasma osmolality (354 ± 11 mOsmol kg-1), appeared similar to values found in HYD frogs at the end of winter conditioning  with levels in frogs frozen to -8 or -16°C exceeding 1300 μmol g-1 dry tissue  in glucose with freezing, but the pattern of variation among groups differed from that seen with liver: glucose concentration in gracilis of the cyclic-freeze frogs was 2.3-fold higher than that found in frogs frozen to -8 or -16°C.Concentration of glucose in liver increased dramatically during freezing  with freezing, as the level in every group of frozen frogs was about two-fold greater than that in unfrozen frogs  among groups.Urea concentration in liver increased (en frogs . In cont-1 dry tissue) in unfrozen frogs, but markedly higher  in frozen frogs. The particular freezing treatment used had no influence on the amount of lactate accumulating in liver, although a significant increase in muscle lactate occurred only in frogs frozen to -16°C , indicating that they did not lose body water or substantially catabolize tissue during the trial.In our freeze tolerance trial, all five of the frogs subjected to cyclic freeze/thaw episodes and ultimately cooled to -8°C exhibited normal behaviors when first examined, ~30 h after thawing was initiated. They remained healthy during an ensuing 7-week period of monitoring. SBM of these frogs determined immediately before freezing (2.82 ± 0.17 g) and after thawing (2.73 ± 0.20 g) did not differ , which r study;  and probR. sylvatica. This contrasts with the case of desert anurans, which heavily draw on lipid stores to fuel metabolism during dormancy [Gluconeogenesis in the pre-hibernal period apparently is driven by the breakdown of lipids to suitable precursors. Our frogs, like those examined previously , cataboldormancy , 18, 19.R. sylvatica. Free amino acids in plasma were 130–150% more abundant in winter-conditioned frogs than in late-summer frogs, the multi-fold increases in various glucogenic amino acids  likely augmenting glycogen synthesis in the pre-hibernal period and possibly even supporting maintenance metabolism during winter. At the same time, catabolism of these compounds produced copious urea, which accrues in response to oliguria, reduced rates of glomerular filtration, and reabsorption in the urinary bladder, and is retained in hibernation for use as metabolic depressant [We found that protein catabolism plays an important, previously unrecognized, role in the hibernal preparations of pressant  and cryopressant . Notablypressant , increaspressant .We monitored changes in energy balance of frogs during winter conditioning to gain insights into the potential role of starvation in the proteolytic response. Complements of crickets consumed by hydrated and dehydrated frogs contained considerable energy , although, overall, these frogs probably expended more energy than they acquired. Their carcasses exhibited reduced concentrations of energy and organic matter, but an apparent increase in nitrogen, which became concentrated in the remaining organic component. However, these changes do not necessarily signify metabolic distress, as some matter and energy were simply transferred from atrophying muscle to glycogen stores in liver, which was excluded from the material being analyzed. Our findings that total carcass energy was not significantly reduced, and that glycogen depots in liver and muscle were enlarged , argue R. sylvatica engages in strenuous mating activity upon hibernal emergence, its potentially adverse effect on muscle performance. They should also elucidate mechanisms underlying the differential responses between R. sylvatica and atrophy-resistant anurans that effectively preserve the mass and mechanical properties of muscles during prolonged hibernation [R. sylvatica potentially stems from changes in endocrine status, as, for example, protein degradation is accelerated by insulin deficiency [Mass of the gastrocnemius decreased by 31–38% during winter conditioning, indicating that skeletal muscle is a principal source of the protein consumed in the pre-hibernal period. Costanzo et al.  observedernation  or estivernation . Extensificiency . Some raficiency , coincidficiency ; such chR. sylvatica from a cool-temperate population (southern Ohio) varied seasonally from 2 to ~50 μmol ml-1, the higher values coinciding with reductions in environmental water potential and body water content. However, a recent study revealed that osmotic stimuli are not essential for frogs to accumulate urea for winter use, as urea reached 29 μmol ml-1 in Ohioan R. sylvatica that remained hydrated during simulated hibernation [-1 herein; 106 μmol ml-1 in Costanzo et al. [-1) when hydrated [The importance of urea in amphibian physiology is best recognized for its role in defending body water during periods of osmotic stress (see review: ). Costanernation . Moreoveo et al. ) freeze-o et al. , 34. IndR. sylvatica, ureagenesis was enhanced by adjustments at the molecular level in liver and skeletal muscle. In hydrated frogs, the pattern of change with winter conditioning in the plasma free amino acid pool, notably an increase in glutamate, alanine, and valine, and a decrease in glutamine, resembles the change in free amino acids occurring in skeletal muscle during estivation of the spadefoot toad, Scaphiopus couchii [R. sylvatica, an apparent exchange of glutamine for glutamate (via the glutaminase reaction) and marked rise in compounds  that readily transfer amine groups to glutamate are coupled with a coordinated, 70% rise in glutamate dehydrogenase activity in muscle [In our pre-hibernal  couchii . In our n muscle . Altogetn muscle .R. sylvatica could also accelerate ureagenesis during winter conditioning by upregulating the ornithine-urea cycle (OUC) by a process similar to that used by some species of estivating frogs [Conceivably, ng frogs –38. Indeng frogs . FurtherR. sylvatica is well adapted to survive dehydration [-1 and even exceeding 0.25 M in one individual. Possibly, Alaskan R. sylvatica undergoing profound dehydration, or smaller, more dehydration-susceptible individuals, could achieve an even higher uremia. Ability to accumulate urea to high levels varies by taxa and generally correlates with habitat preference [R. sylvatica, a mesic woodland species, clearly is exceptional. Indeed, the concentrations of urea found in our frogs are comparable to those reported for euryhaline and xeric-adapted forms undergoing estivation or osmotic stress , but is supported by their relatively low energy:nitrogen ratio. Given that nitrogen is retained during winter conditioning, but is shifted from a high-energy molecule (protein) to a low-energy one (urea), protein degradation is manifest in a drop in energy:nitrogen ratio, which in fact was more pronounced in dehydrated frogs . EnhanceR. sylvatica was not significantly larger than that of hydrated frogs; however, they might have synthesized more glycogen had our experimental dehydration been more protracted or severe. Conceivably, partial dehydration in the pre-hibernal period may benefit these frogs by bolstering both their uremic and glucosic cryoprotectant systems. In temperate regions, dehydration seems likely because the water potential of soil is particularly low in autumn [R. sylvatica hibernates closer to water bodies [Some xeric-adapted frogs upregulate urea synthesis during osmotic stress in part by catabolizing muscle protein , 37, witn autumn . In Inter bodies  but never bodies .-1) can occur in some species, including salt-adaptable and urea-accumulating estivators [R. sylvatica  co-accumulated with urea during winter conditioning; in contrast, conspecifics from a more temperate population, which do not accumulate as much urea, have no osmotic gap [N-oxide (TMAO) and glycerophosphorylcholine (GPC) reportedly vary seasonally in R. sylvatica from the Great Lakes Region of North America, being most abundant in the fall and winter [Plasma osmolality in winter-conditioned frogs were within the lower range of those achieved by hyperuremic, estivating anurans , 18. In tivators . Similaro et al. ), plasmaotic gap . Future otic gap . Anuransotic gap , 43, 49,d winter . TMAO prd winter , 51, 52 d winter , and theR. sylvatica, effectively stabilize proteins and improve cell survival under freeze/thaw stress [In some organisms, winter conditioning or cold acclimation increases the size of the pool of free amino acids . In the w stress , 55, 56.R. sylvatica, corporal freezing is accompanied by an extensive, reversible dehydration of the organs. Physicochemical forces governing the process are not fully understood, although it is clear that organs dehydrate to variable degrees as freezing proceeds, and the translocated water ultimately freezes within the lymph sacs, subdermal spaces, and coelom [In d coelom . Our pred coelom , whereasd coelom . The lat-1 tissue fluid, were computed from mean water and solute concentrations and thus incorporate the effect of organ dehydration during freezing. Actual cryoprotectant levels in frozen tissues would be considerably higher due to freeze-concentration of the solution remaining within them.Values, expressed as μmol mlR. sylvatica critically depends on the capacity to mobilize sufficient amounts of the cryoprotectant glucose during the early stages of freezing. In liver, glycogen is rapidly degraded to glucose, which must be distributed to tissues throughout the body before freezing progresses to the point that circulation ceases. Biochemical control of this glycogenolytic response has been studied extensively in R. sylvatica [-1 [Freezing survival of ylvatica , includiylvatica . Experimtica [-1 . Becausetica [-1 ), we hyptica [-1 ) may be R. sylvatica, corporal freezing reportedly does [R. sylvatica muscle has the enzyme, glucose-6-phosphatase, needed to dephosphorylate glucose-6-phosphate , it seeR. sylvatica frozen to −2.5°C, suggesting that, as with glucose, hepatic synthesis of this agent is further stimulated by somatic freezing [Urea concentration in liver rises in Alaskan (and Ohioan) freezing . Accordifreezing , 7, 61, freezing . Urea’s freezing . It is afreezing . AdditioR. sylvatica accumulates urea primarily in anticipation of freezing and glucose after freezing has begun. Both agents colligatively limit ice formation and cellular dehydration, but each also provides benefits such as antioxidation, metabolic regulation, glycolytic fuel supply, ischemic injury prevention, membrane stabilization, and protein protection and renaturation [turation , 54. Altturation , sustainturation ). Thus, Corporal freezing typically results in cardiac arrest and circulatory failure, and, hence, ischemic hypoxia. Protracted or severe freezing necessitates an even greater reliance on anaerobic glycolysis  and, accR. sylvatica readily recovered from being frozen several times to −4°C before being cooled to −8°C over the course of 19 d. Although freezing adaptations are conventionally studied in subjects given a single bout of experimental freezing, natural fluctuations in ambient temperature can expose organisms to successive cycles of freezing and thawing that potentially could alter their freezing responses and susceptibility to freezing injury. For example, freezing multiple times in quick succession could cause reactive-oxygen species, generated during re-oxygenation of tissues upon thawing, to accrue to levels that overtax anti-oxidation systems [In the present study, Alaskan  systems . In inse systems , 67–70.R. sylvatica, as frogs subjected to multiple freeze/thaw cycles catabolized nearly their entire glycogen depot, probably by resuming glycogenolysis during bouts of refreezing. This superproduction of glucose, coupled with restored circulation during thawing intervals, allowed them to achieve uncharacteristically high levels of cryoprotectant in leg muscle . It appg muscle  and probR. sylvatica experience a series of shallow freeze-thaw cycles in early October, just prior to a deeper, more prolonged freezing exposure, Larson et al. [-1 fresh tissue, only slightly less than that (788 μmol g-1 fresh tissue) determined for frogs experiencing repeated freezes in the field [-1; [Having found that Interior Alaskan n et al.  argued tn et al.  and, furhe field . Nevertheld [-1; ), even eeld [-1; . Neverth


Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects  grouped in discovery and validation independent cohorts. Patients’ urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient’s recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation. Acute kidney injury (AKI) is a multifactorial syndrome, a frequent clinical complication in hospitalized patients and responsible of a substantial morbidity and mortality13Urine represents a combination of both plasma ultrafiltrate and urinary tract proteins, including glomerular filtrated plasma proteins, soluble proteins secreted by tubular epithelial cells and microvesicles as exosomes. Thus, urine composition not only reflects normal kidney function but also contains specific kidney produced proteins which may be altered in response to underlying physiopathologyIn the confirmation cohort, target analysis of urinary RBP4 and KNG1 was performed by liquid chromatography mass spectrometry in selected reaction monitoring mode (SRM-LC-MS/MS) for validation. In this case, individual urine samples were analyzed to assess individual behavior (molecular response). Again, significant differences were observed between control group and AKI patients for RBP4 and KNG1  in the fTo further investigate a potential prognostic value for RBP4, we analyzed individual samples collected from a new randomized cohort of a total of 23 patients and 13 healthy subjects by WB. Thirteen of those 23 patients could be monitored at the three time points. On average, including all patients monitored (and not only those for whom sampling was possible at the three time points), RBP4 increased significantly at T1 and progressively decreased thereafter towards normalization, as expected in view of DIGE and SRM data . Persistde novo or worsening CKD may be equally useful to identify a high risk population that may be enrolled in future clinical trials. In this study, recovery means sCr values ≤1.3 mg/dL by T3 for non-CKD patients of returning to basal levels for CKD individuals, according to clinical criteria. In a further step, patients’ response was analyzed individually in view of their recovery. In daily clinical practice a substantial proportion of AKI patients have underlying CKD (30% in our series) as does a significant proportion of the general population around 10%)%10. ThusThe human urinary proteome was here first investigated in the search for significantly altered molecules in response to AKI31415161718KNG1and RBP4 genes were two of the top kidney up-regulated ones in an end stage renal disease (ESRD) mouse model2124We also found increased urinary levels of RBP4 in response to AKI. The rise was observed from the first 48 h of nephrological diagnosis and maintained at discharge on average, although individual differences were further explored and confirmed related to recovery. This low molecular weight protein is easily filtered by the glomerulus and reabsorbed for the most part in proximal tubules. It has been proposed as the best indicator of tubular dysfunction, as a minor reduction in tubular function may lead to increased RBP4 excretion. In consequence, urinary RBP4 level has been proposed as the most sensitive biomarker for loss of proximal renal tubule function in humans3940The coexistence of common mechanisms in different renal disorders with subjacent complex cellular responses taking place simultaneously make the specific identification of biomarkers of AKI, allograft dysfunction, diabetic renal complications or CKD development a real challenge. Some of the proposed biomarkers in the literature are in fact coincident in various diseasesThe use of panels of biomarkers to the detriment of individual molecules should be explored to improve accuracy and specificity. A specific assay for these RBP4 and KNG1 proteins quantitation, based on SRM methodology, was here developed which enormously facilitates feasibility in the clinic in terms of sensitivity and throughput.One limitation of the study may be the number of recruited subjects, wide distribution for age, diabetes, hypertension or drugs. On the other hand, serum creatinine (sCr) and urinary output are the key clinical tools to define AKI. However, assessment of urinary output is more prone to error, especially in patients without an indwelling bladder catheter44Urinary KNG1 and RBP4 clearly respond to AKI. Additionally, RBP4 could predict recovery by monitoring this protein levels over time after AKI, as RBP4 reflects patient’s normalization earlier than sCr values do. Further evaluation of KNG1 and RBP4 diagnostic and prognostic value should follow (e.g. in a prospective study where their role in earlier diagnosis could be investigated), alone or in combination with other proposed markers.KDIGO criteriaUrine samples from 55 AKI patients without proteinuria, diagnosed by Nephrology Department staff, were collected at Hospital Valdecilla  at three time points: diagnosis within the first 48 h (T1), at 7 days of follow-up (T2) and at Nephrology discharge (T3). AKI was defined and classified according Sample collection procedures were in accordance with the Helsinki declaration and signed informed consent was obtained. Approved consent was obtained from Ethical Committee for Clinical Research from Cantabria , endorsed by IDIVAL.Differential protein analysis was performed by DIGE . A total of 20 AKI patients and 20 control subjects were used in this first discovery phase, to identify strongest protein alterations in urine in response to AKI. Four individual urine samples from each group were pooled, making a total of 5 pools to be analysed per group . 5 nmol CyDye DIGE Fluor labelling kit  was used to label proteins following manufacturer’s instructions. Labelled samples were loaded onto IPG strips  and isoelectric focusing (IEF) was carried out in a PROTEAN IEF CELL (BioRad)http://www.matrixscience.com). In all identified proteins, the probability score was greater than the one fixed by Mascot as being significant, that is, a p value under 0.05.Spots selected for analysis were in-gel reduced, alkylated and digested with trypsin according to Sechi and ChaitAs previously published by our group and others79A different cohort to that used in the discovery phase was recruited for confirmation of candidate markers by WB. 10 mL urine samples were collected from 23 AKI patients and 13 healthy individuals. The same amount of total protein (10 μg) total protein was dissolved in Laemmli buffer and loaded per lane in a 12% acrylamide gel (4% stacking). Membranes were blocked with PBS containing 7.5% non-fat dry milk powder and 0.1% Tween-20 for 1 h at room temperature. Membranes were then incubated for 1 h with specific primary antibody rabbit monoclonal anti-KNG1 (1:5000) (Abcam) or with rabbit monoclonal anti-RBP4 (1:50000) (Abcam) in phosphate buffered saline in Tween-20 (PBS-T) (0.05%) containing 5% non-fat dry milk. Finally, they were incubated with horseradish peroxidase (HRP)-conjugated rabbit TrueBlot® (1:1000) (Rockland) for KNG1 or HRP-conjugated goat anti-rabbit (1:10000) (Nordic) for RBP4 as secondary antibody, in PBS-T (0.05%) containing 5% non-fat dry milk. Detection was performed by enhanced chemiluminescence  following the instructions of the manufacturer. Protein bands were quantified by ImageJ software.Concentration of RBP4 was additionally measured by ELISA in urine from a cohort of 8 control subjects and 8 AKI patients following manufacturer’s instructions .Statistical analyses were performed using SPSS, v15.0 , and GraphPad Prism 6 (version 6.01) software. The Kolmogorov-Smirnov test (KS) was used to assess the normal distribution of clinical variables. Quantitative variables were expressed as mean ± standard deviation (SD) or median and interquartile range (IR) for variables with non-normal distribution. Kruskal-Wallis with Dunn’s multiple comparisons test was calculated and differences were considered significant when p value < 0.05. For receiver operating characteristic (ROC) curve, assessment confidence level was selected as 95% and p value < 0.0001.How to cite this article: Gonzalez-Calero, L. et al. Urinary Kininogen-1 and Retinol binding protein-4 respond to Acute Kidney Injury: predictors of patient prognosis? Sci. Rep.6, 19667; doi: 10.1038/srep19667 (2016).


Paralichthys olivaceus prdm14 (Po-prdm14) gene and isolated the 5' regulatory region of Po-prdm14 by whole-genome sequencing. Peptide sequence alignment, gene structure analysis, and phylogenetic analysis revealed that Po-PRDM14 was homologous to mammalian PRDM14. Results of real-time quantitative polymerase chain reaction amplification (RT-qPCR) and in situ hybridization (ISH) in embryos demonstrated that Po-prdm14 was highly expressed between the morula and late gastrula stages, with its expression peaking in the early gastrula stage. Relatively low expression of Po-prdm14 was observed in the other developmental stages. ISH of gonadal tissues revealed that the transcripts were located in the nucleus of the oocytes in the ovaries but only in the spermatogonia and not the spermatocytes in the testes. We also presume that the Po-prdm14 transcription factor binding sites and their conserved binding region among vertebrates. The combined results suggest that Po-PRDM14 has a conserved function in teleosts and mammals.PRDM14 is a PR (PRDI-BF1-RIZ1 homologous) domain protein with six zinc fingers and essential roles in genome-wide epigenetic reprogramming. This protein is required for the establishment of germ cells and the maintenance of the embryonic stem cell ground state. In this study, we cloned the full-length cDNA and genomic DNA of the  N-terminal PR domain and several C-terminal Krüppel-type zinc finger motifs. The PR domain was first reported as the PRDI-BF1-RIZ1 homologous domain [prdm genes expanded in vertebrates and prdm7/prdm9 further duplicated in primates. The PRDM family in primate genomes consists of 17 putative orthologs (such that a single rodent co-ortholog corresponds to the primate pair formed by PRDM7 and PRDM9). By contrast, the PRDM family in rodents, birds, and amphibians contains 16 putative orthologs. Meanwhile, researchers only detected 15 orthologs in fish because they could not identify the PRDM17 ortholog, which was possibly because of the genome assembly used [PRDM (PRDI-BF1-RIZ1 homologous domain-containing) family members are characterized on the basis of a conserved s domain ,3. To das domain . The adds domain ,6,7,8. Ps domain ,12,13,14s domain . PreviouN-terminal PR domain and six Krüppel-type zinc finger motifs. The PR domain and zinc fingers are highly conserved among vertebrates. In general, PRDM14 recruits genomic targets through the zinc finger domains. Previous studies identified that the PRDM14-bound regions contain the unique non-repetitive sequence 5'-GGTCTCTAA-3' in humans and mice [As a PRDM family member, PRDM14 has an and mice ,18. Thisand mice ,20. Otheand mice ,18. In aand mice ,21. Moreand mice ,18,21. Aand mice ,21,22,23and mice , and theand mice ,26,27. Pand mice . The funParalichthys olivaceus) is an important farmed marine fish, which shows morphological left-right asymmetry, such as one eye migrates to the opposite side (metamorphosis) during the larval stages. Similar to mammalian ESCs, fish ESCs can replicate indefinitely and differentiate into all three layers of the embryo. To date, some fish ESC lines have been established under a feeder-free condition from blastula-stage embryos [Po-prdm14 gene is critical.The Japanese flounder  and has a 214 bp 5' UTR, a 576 bp 3' UTR, and a 1833 bp open reading frame (ORF)  and Danio rerio prdm14 (Dr-prdm14). In general, the 5' UTR sequences are located on the first exon and on part of the second exon.The full-length cDNA sequence of me (ORF) . Po-PRDM 235–352 . A compane exons , which dPo-PRDM14 and other known PRDM14 orthologs was performed using ClustalX2.1 ; POU domain, class 5 transcription factor 1 (Oct3/4); the composite binding site for Oct3/4, Sox2, Nanog, Tcf3 (Tcf7l1), and Sall4b in pluripotent cells (ONST); SMAD1/5; PR domain containing 1 (PRDM1); activator protein-1 (AP-1); and specificity protein 1 (SP-1)  A. A TATA1 (SP-1) B.Po-prdm14 promoter sequence had a higher similarity to those of sea bass (39%) and fugu (32%) than to those of other vertebrates. A total of 23 putative conserved regions were identified in the alignment of teleost homologs. One region was also conserved in the similar upstream region of the mouse homolog, whereas four regions were conserved among humans, chimpanzees and teleosts, but not rodents.Considering that the TSSs of some species were not known, we had to designate the translational initiation site (ATG) as +1 C. The PoPo-prdm14 mRNA-positive signals were almost undetectable before the morula stage. As embryonic development progressed, the amount of Po-prdm14 transcripts rapidly increased, and peaked at early gastrula stage before they suddenly decreased from the mid-gastrula stage onward. The amount of Po-prdm14 transcripts were greatly reduced at the end of the gastrulation stage. Subsequently, the Po-prdm14 transcripts were maintained at very low levels, with only a slight increase during the hatching stage  to be a cytosine (C) residue located 85 bp upstream from the start codon ATG. Consensus basal core promoter elements, such as the TATA-box, GC-box and CAAT-box, were assumed in their expected positions; these elements might comprise the core promoter of Po-prdm14 , OSNT , NANOG (homeobox transcription factor NANOG), and FOXP1_ES . Interestingly, PRDM14 co-localized with OCT3/4, SOX2, NANOG, and P300 [Po-nanog was maternally inherited, expressed highly before gastrula and expressed specific in gonads among adult tissues. The analysis in Po-nanog showed potential Po-PRDM14 binding sites on Po-nanog promoter [Po-prdm14 promoter, which suggests that Po-NANOG and Po-PRDM14 might interact with each other to some extent.and P300 ,21 and band P300 ,21. A prpromoter . In thisB. Motif composed of putative binding sites for PGC-related or sexual development. Such predicted binding sites include DMRT3 (doublesex and mab-3-related transcription factor 3), SPZ1 , HBP1 (HMG box-containing protein 1), SOX/SRY-sex/testis determining and other related HMG box factors: SOX5 (SRY-related HMG-box gene 5), SOX9 (SRY-related HMG-box gene 9), and PRDM1 (PR domain containing 1). The specification of PGCs occurs through the integration of three key events: repression of the somatic program, reacquisition of potential pluripotency, and the ensuing genome-wide epigenetic reprogramming; PRDM14 is critical to the last two events in mouse [in mouse ,49,50,51C. Motif composed of putative binding sites for regulation. Such prognostic binding sites include AP-1 (activator protein 1), SP-1 (specificity protein 1), CREB (cAMP-responsive element binding proteins), and GATA binding factors .Po-prdm14 promoters and other vertebrates predicted that some binding sites, such as DMRT3, PRDM1, OSNT, and SOX9, were conserved among vertebrates, particularly among teleosts  of blastocysts, the early epiblast, and mESCs [prdm14 expression starts from the 8-cell stage and continues in the ICM [prdm14 is expressed in hESCs, which are similar to mESCs [The expression of  tissues . Therefot stages . RT-qPCRin mouse . In mousnd mESCs ,26,48,52 the ICM ,53,54. Mto mESCs ,55,56.Po-prdm14 mRNA-positive signals were the strongest in the early-gastrula and present in all blastomeres. While in zebrafish, Dr-prdm14 gene was maternally inherited. Sun et al. displayed the expression levels of Dr-prdm14 in embryos at 2-cell stage, 64-cell stage and blastula stage with WISH analysis, and the result revealed high Dr-prdm14 expression in these stages [et al. via WISH showed that Dr-prdm14 expressed in early-gastrula, late-gastrula, bud stage, and in neuron precursors among somite stages [Dr-prdm14 might express in almost all stages through zebrafish development and had important roles in the neural development in zebrafish. However, the expression pattern of Po-prdm14 and Mus musculus (Mm-prdm14) differed from that of Dr-prdm14, as they specifically expressed in several stages. But through WISH technology we did not detect the Po-prdm14 in neural expression. One possibility might be the functions of PRDM14 homologues were not strictly conserved despite their conserved protein sequences.Whole-mount in situ hybridization (WISH) showed that positive signals almost coincided with the RT-qPCR results . In addie stages . Liu et e stages . Based oPo-prdm14 gene was expressed in the nucleus of oocytes  samples were obtained from a commercial hatchery in Yantai, Shandong province, China. Fertilized eggs were obtained by artificial fertilization and were incubated at 17 ± 1 °C in sterile sea water with an open recirculation water system and sufficient air supply. The different embryonic stages were observed under a stereomicroscope. Three pools of samples at embryonic stage  and post-embryonic stages , were separately collected from mixed families with a nylon net (100-mesh). Embryos and larvae were immersed in 1 mL of RNAwait liquid  overnight at 4 °C and then stored at −80 °C until further use. The staged embryos and gonads (ovary and testis) for ISH were washed twice by PBS (in DEPC) and then fixed in 4% paraformaldehyde in PBS overnight at 4 °C. After removing the envelopes, the embryos were dehydrated in a gradient series of increasing methanol concentration and stored in 100% methanol at −20 °C. Animal experiments were performed according to the Regulations for the Administration of Affairs Concerning Experimental Animals . The procedures were also approved by College of Marine Life, Ocean University of China .Japanese flounder  following the manufacturer’s instructions. Genomic DNA was extracted from embryos via the phenol–chloroform method, precipitated with isopropanol, washed with 70% ethanol and 100% ethanol, and then dissolved in TE buffer [Total RNA was separately isolated from the embryos using Trizol reagent  in accordance the manufacturer’s instructions . The extE buffer . The quaDicentrarchus labrax prdm14 (Dl-prdm14) gene was obtained from the whole-genome sequence of P. olivaceus as constructed by our laboratory (unpublished). After a BLAST query of both sequences on Spidey (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/), the general distribution of introns and exons in Po-prdm14 gene was acquired and found to be similar to those of other known species. From this information, two pairs of primers (prdm14-core1 and prdm14-core2). These partial fragments had an overlap that could aid the assembly of the larger central region of the full-length Po-prdm14 gene. The remaining unknown cDNA region was sequenced by 5' and 3' rapid amplification of cDNA ends (RACE) reactions with the SMART™ RACE cDNA Amplification kit  in accordance with the manufacturer’s protocol with RACE primers  (prdm14-full long) were designed to verify the complete open reading frame (ORF). The genomic DNA sequence was obtained via amplification with three pairs of primers .A scaffold with the highest identity with the  primers  were des and -3) . On the Po-prdm14 gene was found in the P. olivaceus genomic data. After a BLAST query in the GenBank database, the 1929 bp region in this sequence was predicted to be the Po-prdm14 promoter sequence, whereas the other region belonged to the Ncoa2 gene. A pair of specific primers (prdm14-promoter) was designed to verify the promoter sequence. All the amplified PCR products were separated by 1.5% agarose gel electrophoresis, purified with the Zymoclean Gel DNA Recovery kit, and then cloned into the PMD18-T vector . Positive clones were screened by specific-primers and sequenced.A 4 kb upstream sequence in the prdm14-RT). A pre-experiment was performed to confirm the specificity of the cDNA PCR product. RT-qPCR was performed with a LightCycler480 apparatus in a 20 μL reaction volume containing 1× SYBR® Premix Ex Taq™ II , 0.2 μM of each primer, and 1 μL of dilute cDNA (10 ng/μL). The PCR profile used was 95 °C (5 min) for pre-incubation, followed by 45 cycles at 95 °C (15 s) and 60 °C (45 s). Melting curves were generated at the end of each run to verify the presence of a single product. A negative control (no templates) was included for the PCR run. The 18S ribosomal RNA (rRNA) (18S-RT) and the ubiquitin conjugating enzyme (UbcE) (UbcE-RT) sequences were selected to normalize the target gene levels via the standard curve method [p < 0.05 were considered statistically significant. All data were expressed as the mean ± standard error of the mean (SEM).Primers for RT-qPCR were designed ; these pe method ,60. All Po-prdm14 embryos and tissues were analyzed by ISH with a 641 bp probe spanning the C-terminal coding region and the 3' UTR of the Po-prdm14 cDNA (prdm14-ISH). DIG-labeled RNA sense and anti-sense probes were synthesized with a DIG RNA Labeling kit  in accordance with the manufacturer’s instructions. The ISH of whole embryos and tissues was performed as described by Gao et al. [The o et al. .http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the EMBL Uniprot (http://www.uniprot.org/) database. Multiple sequence alignment of Po-PRDM14 and the PRDM14 sequences of other species was performed with ClustalX2.1. To evaluate the evolutionary relationship between the predicted Po-PRDM14 protein and other vertebrate PRDM14, we constructed a phylogenetic tree from the full-length peptide sequences with MEGA using the Poisson correction distance via the neighbor-joining method with 1000 bootstrap replicates. The PRDM14 sequence of Caenorhabditis elegans served as the outgroup. The conserved domains were predicted using the simple modular architecture research tool (SMART http://smart.embl-heidelberg.de/) [http://www.ebi.ac.uk/interpro/search/sequence-search) of the amino acid sequence of Po-PRDM14. Neural Network Promoter Prediction  was used to predict the promoter element of Po-prdm14. Bioinformatics analyses of the promoter sequence and potential TF binding sites within the 5' regulatory region of Po-prdm14 were performed with the online program MatInspector (http://www.genomatix.de/matinspector.html) [http://www.cbrc.jp/research/db/TFSEARCH.html), Match 1.0 (http://www.gene-regulation.com/cgi-bin/pub/programs/match/bin/match.cgi), Alibaba2.1 , and TFBIND (http://tfbind.hgc.jp/) [http://meme.nbcr.net/meme/cgi-bin/meme.cgi) and Dialign software from the Genomatix suite  were used to identify the conserved DNA motifs in the 5' regulatory region of Po-PRDM14 and other vertebrates.Homologous protein sequences were obtained through a BLAST search of the NCBI GenBank (erg.de/) ,62, and or.html) , combinehgc.jp/) . The Mul


This article contains schemes, original experimental data and figures for an integrated modularized microfluidic system described in “An integrated microfluidic system for bovine DNA purification and digital PCR detection  Specifications TableValue of the data•The structure and fabrication details of an integrated modularized microfluidic system are given.•Amplification plots and DNA yields resulting from on-chip isolation can be compared with the benchtop procedure.•Data from qPCR were compared with data from dPCR.11.1Integration is increasingly recognized as an important technical challenge in lab-on-a-chip device. More efficient and lower cost integrated microfluidic systems decrease or eliminate reliance on traditional lab equipment. Chin et al. We have integrated a NA extraction module and a dPCR reaction module on this chip. As shown in For the DNA isolation form tissue, we introduced 70% (v/v) ethanol, 70% (v/v) ethanol, buffer MP3, and lysate orderly by adjusting the knob of pipettor. As shown in Fluidic isolation of the NA extraction region and dPCR region was essential for the purposes of avoiding the incompatibility of the NA extraction with the amplification process. Because of the pull force from the negative pressure from the other side of the capture zone, the reagents could not seep into the channel that connected the dPCR module and NA extraction module, in case the reagents disturbed the subsequent dPCR reaction. As shown in 1.2.The purification of NA from specimens was laborious and time-consuming until BOOM et alWhen 20 μL lysed sample with the Mag-Bind particles was pulled into the chip and passed through the capture zone by the negative pressure from other side, the Mag-Bind particles with NAs were collected by a magnet. Then the three sections of reagent in Teflon tube were pulled into the capture zone and washed away proteins and/or other contaminants on the Mag-Bind particles. Finally, DNA was eluted by PCR mix and introduced into the dPCR region for subsequent dPCR analysis.U statistical test  (But when the Mag-Bind particles were collected together and washed by 70% (v/v) ethanol, the Mag-Bind particles were clumped together and resisted washing and elution. We introduced a groove for magnet sliding above the NA capture zone to drive the magnetic particles movement around and improve the efficiency of washing and elution. The elution efficiency of PCR mix in a 0.2 mL tube and microdevice was determined by gel electrophoresis . The gen02>0.05) . The dat02>0.05) . The bov02>0.05) .The authors declare that they have no competing financial interests.


Chemokines such as MIP-3β/CCL19 are important factors in the mechanism of cell migration and pathogenesis of central nervous system (CNS) inflammatory reactions. The hypothesis of this study is that CCL19, also known as MIP-3β, is involved in the pathogenesis of inflammatory and non-inflammatory CNS diseases of dogs. Experiments were performed on cerebrospinal fluid (CSF) and serum samples of dogs affected with steroid responsive meningitis-arteritis (SRMA) during the acute phase as well as during treatment. Dogs with SRMA were compared to dogs with presumed meningoencephalomyelitis of unknown origin (MUO), and both groups sub-categorized into dogs receiving no therapy and with patients receiving prednisolone therapy. Idiopathic epilepsy (IE), a group with normal CSF cell count, was used as a control. Additionally, dogs with intervertebral disc disease (IVDD) of varying severity were analyzed. Chemokine concentrations were determined by enzyme linked immunosorbent assay. Migration assays were performed on seven selected CSF samples using a disposable 96-well chemotaxis chamber.CCL19 was detectable in CSF samples of all dogs. Dogs with untreated SRMA and MUO displayed pronounced CCL19 elevations compared to the control group and patients receiving glucocorticosteroid treatment. CSF cell counts of untreated SRMA and MUO patients were significantly positively correlated with the CCL19 CSF concentration. IVDD patients also had elevated CCL19 concentration compared to controls, but values were considerably lower than in inflammatory CNS diseases. Selected CSF samples displayed chemotactic activity for mononuclear cells in the migration assay.CCL19 CSF concentrations were markedly elevated in patients affected with the neuroinflammatory diseases SRMA and MUO and showed a strong correlation with the CSF cell count. This chemokine may play an important role in the pathogenesis of SRMA and MUO. The elevation of CSF CCL19 in IVDD suggests that it may also be involved in the secondary wave of spinal cord injuries. In several neurologic diseases, a pleocytosis is often detected in the cerebrospinal fluid (CSF). Lymphocytes and plasma cells have been shown to prevail in the cell count in viral infections , chronicA total of 141 patients were analyzed following referral to the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany and the Veterinary Emergency and Specialty Center (VESC) Santa Fe, NM, USA . The study was approved by the ethics committee of the University for Veterinary Medicine Hannover. The experimental protocols and procedures in healthy dogs were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the authorities in Lower Saxony . CSF and serum samples for CCL19 assays were obtained and stored at -20°C. Blood was collected via cephalic, saphenous or jugular venipuncture into serum tubes. CSF collection was performed under general anesthesia by cisternal puncture for SRMA, MUO, IE and healthy dogs or lumbar puncture for IVDD dogs. The CSF was centrifuged and the sediment was removed. The remaining CSF was stored at -20°C within 2 hours of sample collection. Number of patients and their laboratory findings are provided in Table SRMA group: Fifty one patients were diagnosed with SRMA based on clinical signs, complete blood and CSF examinations, normal cervical radiographs, elevated IgA levels in CSF and serum, response to glucocorticosteroid treatment. The untreated SRMA group consisted of 25 patients which received no medication at the time of sample collection. Nine of these dogs were evaluated separately, five patients having received a one-time pre-treatment with corticosteroids by the referring veterinarian and four patients having relapsed after stopping previous therapy. CSF and blood findings are summarized in Table MUO group: CSF was also obtained from 27 patients with presumed MUO. Sixteen of these patients received no therapy (untreated group), while 11 patients were already being treated with prednisolone (treated group)   were anaHealthy dogs: Additionally, six samples were obtained from healthy dogs Table  . An ELISA Kit specific for canine CCL19 (E90096Ca 96 Tests) was used according to manufacturer’s instructions . Briefly, the microtiter plate provided in the ELISA kit has been pre-coated with a monoclonal antibody specific to CCL19. CSF samples were used undiluted or were diluted to a dilution of 1:13 with the standard diluent depending on the CCL19 output concentration. Serum samples were used undiluted for the ELISA. Seven wells were prepared for the dilutions of standard, one well was left blank and the rest of the 96 wells were used for the CSF and serum samples. Four CSF and serum samples of the IE group served as a control and one of these IE samples was pipetted to the plates in each experiment. Samples as a positive control were CSF and serum samples of the neuroinflammatory group and the same sample was used in each experiment at same dilution. The color change was measured spectrophotometrically at a wavelength of 450 nm ± 10 nm on a microplate reader equipped with the analysis software Gen 5 . The detection range of this method was 15.6 - 1000 pg/ml. The standard curve was created using a lyophilized standard reagent  provided in the ELISA kit. Standard concentrations used for the ELISA were 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml. The minimum detectable dose of canine CCL19 is 6.5 pg/ml. All measurements of the CSF and serum were performed in duplicates.® RPMI Media 1640) containing 1% bovine serum albumin (BSA) and a negative control of pure medium. The loaded chamber with added cell suspension on the top side of the filter was cultured at 37°C in humidified air with 5% CO2 in an incubator  while the migration time for the cell suspension was measured at 30 minutes, 1 hour, 1.5 hours and 2 hours. The filter was removed and cells that had migrated through the filter to the lower wells were counted by alamarBlue® assay . AlamarBlue® reagent was added as 10% of the sample volume, followed by 1 to 3 hours incubation at 37°C. Resazurin, the active ingredient of alamarBlue® reagent, is a non-toxic, cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced by viable cells to resorufin, a compound that is red in color and highly fluorescent. The resulting fluorescence was read on a microplate reader equipped with the analysis software Gen 5 .To evaluate chemotactic activity of measured CSF samples for mononuclear cell migration, assays were performed using a disposable 96-well chemotaxis chamber with polycarbonate filters  . CSF samples of seven patients were examined, three untreated patients diagnosed with SRMA, two untreated patients with the suspected diagnosis of MUO and two patients with IVDD. Peripheral mononuclear cells (PMCs) were separated from five milliliters of fresh EDTA blood collected via cephalic venipuncture of a healthy dog. PMCs were separated by Ficoll gradient centrifugation according to the technique described by Toth et al. 1992 . PMCs weFor group comparison, the Kruskal-Wallis test and the Mann–Whitney–Wilcoxon tests were used. Using the Shapiro-Wilk test, an abnormal data distribution was found. Correlation between two variables were tested by Spearman and Kendall rank correlation tests. Statistical significance was set at the 5% level (P < 0.05).CCL19 was measurable in all 21 samples of patients with idiopathic epilepsy and in the 6 samples from healthy donors (Table Significantly elevated CCL19 concentrations were observed in CSF samples of untreated SRMA and MUO patients compared to the control group (IE)  as shown in Figure The CSF CCL19 concentrations of both untreated SRMA and untreated MUO patients were significantly correlated with the CSF cell count .SRMA and MUO patients under glucocorticosteroid treatment displayed no correlation between CSF cell count and CCL19 concentration as indicated by the results of Spearman and Kendall’s rank test . The CSF CCL19 and serum CCL19 concentrations did not correlate with each other indicating that CSF elevation is not due, at least entirely, to BBB disruption . Total protein levels within the CSF from untreated SRMA dogs had a weak correlation with CSF CCL19 concentrations using Spearman rank test ; the Kendall rank test was performed due to the occurrence of outliers and revealed a significant correlation . Dogs with MUO that did not receive therapy had no strong correlation between CCL19 CSF and CSF protein concentration .CSF CCL19 concentrations of IVDD are significantly elevated compared to the control group (p <0.05) as illustrated in the Box-and-Whisker Plots in Figure No significant correlation of CSF CCL19 concentrations to CSF cell count was found among the IVDD group . CCL19 CSF concentration and CCL19 serum concentration were significantly correlated within IVDD group 2/3  while IVDD group 4/5 had no significant correlation .CSF samples were used to induce mononuclear cell chemotactic activity. CSF of neuroinflammatory diseases, including SRMA and MUO, showed high cell migration as illustrated in Figure The results of this study support the hypothesis that the protein CCL19 is elevated in selected CNS diseases and might be involved in the pathogenesis of mononuclear cells migration into the subarachnoid space of dogs with inflammatory CNS diseases and non-inflammatory CNS diseases such as intervertebral disc disease (IVDD). CSF levels of the chemokine CCL19 were analyzed in dogs afflicted with SRMA, an inflammatory CNS disease, and compared to other neuroinflammatory diseases (MUO), traumatic IVDD, a non-inflammatory CNS disease, idiopathic epilepsy (IE) and normal unafflicted animals.t al.[t al. [et al.’s study revealed that a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells exists [et al. also observed significant levels of CCL19 in patients affected with MS and patients affected with other neuroinflammatory diseases and suspect involvement in T-cell trafficking [CCL19 was detectable in healthy dogs and in patients with idiopathic epilepsy, a non-inflammatory neurologic disease, which is in agreement with the results of other studies ,16 in tht al. and Pashl.[t al. . The higs exists . Activats exists . Observas exists . Neutrops exists . The neus exists . An uncos exists . Investis exists . Guinti fficking . In our et al.’s study did not reveal that MS patients receiving treatment differ from untreated patients [SRMA and MUO patients receiving glucocorticosteroids had significantly less CCL19 concentrations, showing that steroids have a strong impact in reducing the inflammatory pathway. Krumbholz patients . Glucocopatients  and may et al. suggested CSF pleocytosis is positively associated with the severity of thoracolumbar spinal cord damage in dogs with IVDD. They found that the percentage of CSF macrophages can be used as a prognostic indicator for regaining ambulation in dogs that have lost deep pain sensation [The elevation of CCL19 in IVDD CSF suggests a process of a secondary inflammatory response within the CNS . Gray anensation . The curet al. showed that the expression of CCL19 occurs at the BBB in experimental autoimmune encephalomyelitis and concluded that this chemokine may contribute to T -cell attraction across the BBB [et al. observed in his study that interleukin (IL)-6, IL-8, tau, S100b and glial fibrillary acidic protein were elevated depending on severity of the initial injury. Evaluating this group of inflammatory cytokines, a biochemical model was developed enabling veterinary scientists to predict the ASIA  grade in 89% of their subjects. It was found that the pattern of protein expression within the CSF over the first few days following injury were strong indicators of neurologic outcome and thus, are now a focus of therapeutic research for SCI [The performed chemotaxis assay from CSF samples attracted mononuclear cells. CSF of neuro-inflammatory diseases showed strong chemotactic activity in comparison to the controls. Even CSF samples of dogs with IVDD with a much lower mean CCL19 concentration showed a pronounced impact on cell migration, which suggests the presents of a functional chemotactic protein. Alt  the BBB . CSF con the BBB . CNS res the BBB . Additio for SCI . Using CIn conclusion, CCL19 CSF concentrations were markedly elevated in patients with neuroinflammatory disease and may play an important role in their pathogenesis with recruitment of inflammatory cells into the subarachnoid space of dogs. This study also showed an elevated CSF CCL19 concentration in dogs with IVDD, indicating that the chemokine might also be associated with the secondary wave of SCI, following an acute cord insult. A marked chemotactic activity for mononuclear cells was observed in selected CSF samples of the neuroinflammatory and non-neuroinflammatory disease. Further functional studies could confirm that CCL19 has a major role of mononuclear cell migration into the subarachnoid space of dogs affected with SRMA, MUO, IVDD and may be a precious target for developing new treatment schemes.ASIA: American spinal injury association; BBB: Blood brain barrier; BSA: Bovines serum albumin; CCL19: C-C motif ligand 19; CCR7: C-C chemokine receptor; CNS: Central nervous system; CSF: Cerebrospinal fluid; DC: Dendritic cells; EAE: Chronic experimental autoimmune encephalomyelitis; FLAIR: “Fluid attenuated inversion recovery”; GME: Granulomatous meningoencephalomyelitis; ELISA: Enzyme linked immunosorbent assay; HBSS: Hank’s balanced salt solution; IE: Idiopathic epilepsy; Ig: Immunoglobulin; IL: Interleukin; IVDD: Intervertebral disc disease; log: Logarithmus; MIP-3β: Macrophage inflammatory protein-3beta; ml: Milliliter; MRI: Magnetic resonance imaging; MUO: Meningoencephalomyelitis of unknown origin; μl: microL, microliter; mg: Milligram; ml: Milliliter; NE: Necrotizing encephalitides; No: Number; PBS: Phosphate buffered saline; PMC: Peripheral mononuclear cells; pg: Picogram; RPMI: Rosewell park memorial institute; SCI: Spinal cord injuries; SRMA: Steroid responsive meningitis-arteritis; TMB: 3,3′,5,5′-Tetramethylbenzidine; VESC: Veterinary Emergency and Specialty Center.The authors declare that they have no competing interests.JB designed and performed the ELISA assay experiments as well as the chemotaxis assay experiments and drafted the manuscript. BD participated in the analysis of the study and helped to draft the manuscript. SJS provided the samples and participated in the coordination of the study. LB performed the statistical analysis. RC helped with the execution of laboratory methods. AT conceived of the study, participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown.in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression.Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis. Up to thirty percent of breast cancer patients with metastatic disease suffer from brain metastasis, resulting in very unfavorable prognosis even in comparison to metastatic disease in other sites . To makelinear progression model of metastasis, were a successful treatment of the primary tumor should inhibit seeding to distant organs [This is partially due to the hypothesis of the t organs . Consistparallel progression or early dissemination model) [It is now accepted that in most cases the distant organs are already seeded at the time when the primary tumor is diagnosed (n model) . Thus, en model) . Followin model) –10. In on model) . Recentln model) , 13. Impn model) . For exan model) . Howevern model) . Thus, mop/op mice [op/op model MCs are absent, but microglia are still present, although with reduced numbers [op/op as well as in CSF-1 overexpressing mice [Colony stimulated factor-1 (CSF-1) could serve as a possible target, known to be part of a paracrine loop between carcinoma cells and blood-derived macrophages (MCs) . This pa/op mice . In the  numbers . Howevering mice –21.This astonishing discordant observation of MCs and microglia could be explained by current findings: Resident microglia were shown to be derived from yolk sac progenitor cells during embryonic development, even before the formation of hematopoietic stem cells , 23. TheIn terms of metastasis, these findings have severe consequences because the most affected metastatic organs possess these specialized resident macrophage populations, such as Kupffer cells (in liver), alveolar macrophages (in lung) and microglia (in brain). Directly following extravasation, disseminated metastatic tumor cells encounter these unique resident macrophages whose main task is the protection and homeostasis of their respective organs (organ-specific defense systems). Additionally, these macrophage populations do not only differ from MCs, they also demonstrate distinct organ-specific characteristics. However, the functional interactions of metastatic cells with alveolar macrophages, Kupffer cells or microglia are largely unknown.parallel progression model and the description of these unique macrophage populations led us to focus on microglia during cerebral metastasis to identify possible therapeutic targets and to prevent metastatic colonization. In view of the CSF-1 paracrine loop and its effects on MCs in the primary tumor, we set out to evaluate whether CSF-1 could be a therapeutic target during colonization of the brain parenchyma.The Here we show that microglia are significantly different from MCs, in particular as to their CSF-1 dependency. Further, the expression of the alternative ligand IL-34 is organ specific and carcinoma cells produce significant amounts of CSF-1 as well as IL-34, which partially interferes with the anti-CSF-1 treatment effects.In this study we sought to analyze the effects of an anti-CSF-1 antibody (clone 5A1) on MC-induced invasiveness of breast cancer cells. To this end, we first determined a concentration of 5A1, which affected the MCs but did not influence the breast carcinoma cells. The human breast cancer cell lines MCF-7 and MDA-MB231 were treated with increasing concentrations of 5A1 for 96 h followed by analysis of metabolic cell activity by MTT-conversion. Both cell lines did not show a reduction in their metabolic activity even at the highest concentration tested Figure . In lineCSF-1 is an essential growth factor during the differentiation of myeloid progenitor cells. We thus speculated that MCs would be more sensitive to depletion of CSF-1. To address this question we treated MCs and microglia (MG), with increasing concentrations of the anti-CSF-1 antibody and determined the rate of cell proliferation using the xCELLigence system. As expected, proliferation of MCs was inhibited already at the lowest antibody concentration tested Figure . In contRecently it was shown that IL-34 is an alternative ligand for CSF-1R . We therWe next asked whether the varying effect of 5A1 on the viability of MCs and MG can be further explained by impairing the downstream signaling of CSF-1R, which is mediated by AKT and S6. Therefore, we analyzed the expression of these two proteins and their activated (phosphorylated) forms pAKT and pS6 in MCs and MG in the presence or absence of 5A1 by western blot. Results revealed that the anti-CSF-1 antibody does not alter the expression of AKT and S6 whereas the activation of AKT and S6 is inhibited by 5A1 in MCs but not in MG Figure . This fiIn our previous studies, we demonstrated that MCs induce breast cancer cell invasion , 27. FurIn parallel, we tested the effects on MG-induced invasion  under thBased on our finding that viability of MG was not affected by 5A1, and the fact that expression of the alternative growth factor IL-34 was detectable in brain but not in bone marrow, we next determined whether the toxic effect of 5A1 on MCs could be rescued by CSF-1 or IL-34. Accordingly, we first analyzed the metabolic activity of MCs in co-culture with carcinoma cells as well as after treatment with IL-34. Interestingly, while co-culture with MCF-7 cells did not alter the metabolic activity of MCs, co-cultivation with MDA-MB231 or 410.4, as well as treatment with IL-34 significantly increased the metabolic activity of macrophages Figure , and furIn order to validate the relevance of our findings in human disease, and to test whether CSF-1 depletion could potentially be used for the prevention or treatment of brain metastasis, we examined the expression of the growth factors CSF-1 and IL-34 and the corresponding receptor CSF-1R in brain metastases of cancer patients. We performed qRT-PCR analysis on five brain metastases from lung cancer patients and three brain metastases from breast cancer patients. Analysis of the results revealed that CSF-1, IL-34 and CSF-1R were expressed in all samples although the expression levels for a distinct gene varied. For CSF-1 the difference between the sample with the lowest expression and the sample showing the highest expression was 8-fold. For CSF-1R and IL-34 this difference was 64-fold and 32-fold, respectively Figure . Differeclaudin-low breast carcinoma cells mediated via an autocrine activation of CSF-1R signalling [To further underline the significance of the above findings in a larger dataset we investigated the gene expression of CSF-1, CSF-1R and IL-34 in a cohort of brain metastases of already published microarray gene sets  and compared them for differential gene expression of CSF-1, CSF-1R and IL-34. These analyses revealed CSF-1 to be predominantly expressed in lung brain metastases and to a lesser extend in brain metastases arising from breast cancer Figure . In contgnalling . Thus, wHere we demonstrate that resident MG are not addicted to CSF-1 while MCs are very sensitive to anti-CSF-1 treatment. Furthermore, breast cancer cells are not only a source of CSF-1 as already described, but also a source for IL-34, the alternative ligand for the CSF-1R. This is reflected by its high expression in the metastatic basal-like MDA-MB231 breast cancer cells and in human brain metastases. The great importance of this alternative ligand is underlined not only by the finding that the more aggressive and IL-34-expressing MDA-MB231 cells induced a significant increase in MC proliferation, but also by the fact that IL-34 could partially overcome the effect of anti-CSF-1 on MCs. When analysing IL-34 expression in the bone marrow, as compared with the brain parenchyma, we only detected IL-34 expression in the brain, indicating significant differences in the brain microenvironment  specific mechanism that contributes to resistance to CSF-1 depletion e.g. in the CSF-1 numbers . When emHowever, although we detected no differences in the viability of MG after anti-CSF-1 treatment, MG-induced invasion of breast cancer cells was reduced following anti-CSF-1 treatment, but to a lower extent than MC-induced invasion. These results could point to a phenotypic shift of MG induced by CSF-1 inhibition as already mentioned , 20. A cin vitro systems were applied, we demonstrate that this could have significant influence on innovative treatment strategies addressing the MPS, in particular in metastasis. Additionally, our findings emphasize the role of resident macrophages and organ-specific defense systems during the crucial step of metastasis, at least in metastatic organs with unique macrophage populations, like liver, lung and brain. Importantly, once assuming the parallel progression model of metastasis, organ-specific defense systems represent one of the most important targets to prevent metastatic outgrowth of disseminated carcinoma cells. Thus, strengthening the organ-specific defense systems may be a very potent therapeutic strategy to deplete incipient metastatic cells at distant organs during the treatment of primary carcinomas.Taken together, our most significant finding is the fundamental difference between MCs and MG, supporting the importance of the new MPS classification and the categorization into macrophages, MCs and DCs . AlthougIf not indicated otherwise, all substances were purchased from Sigma . The human breast cancer cell lines MCF-7 and MDA-MB231 were obtained from the American Type Culture Collection . The human cell lines were cultivated in RPMI-1640 medium  supplemented with 10% heat inactivated fetal bovine serum . The BALB/C murine breast cancer cell line 410.4, obtained from the American Type Culture Collection , was cultured in DMEM + 10% FCS. The anti-CSF-1 antibody was obtained from Novartis . Recombinant IL-34 was obtained from BioLegend  and a concentration of 100 ng/ml was used in the experiments.Murine MCs were isolated as described previously . BrieflyPrimary MG cell cultures from newborn (P0) NMRI mice were prepared and cultured as previously described . After 14 MCs per well were seeded in 500 μl medium and 1 × 105 tumor cells were seeded in a transwell insert  containing 500 μl medium. After at least 1 h of incubation, to allow the cells to attach, the transwell insert was transferred into the well containing MCs. Co-cultures were performed for 96 h, before transwells were discarded and MTT measurement was performed for MCs.Cell metabolism was analysed by measurement of MTT  conversion according to standard procedures. Briefly, cells were incubated with 0,5 mg/ml MTT for 4 h at 37°C. Subsequently cells were lysed (5% formic acid in isopropanol + DMSO (2:1)) and optical density was measured at 550 nm. Cells were treated with anti-CSF-1 (5A1) for 96 h before measuring MTT reduction. For co-culture assays 5 × 103 (MDA-MB231), 4 × 104  or 8 × 104 MG cells per well were plated and proliferation/morphology was analyzed for 48 h in quadruplets. Treatment with 5A1 was performed once at time point 0 h.Cell proliferation assays were performed using the xCELLigence RTCA DP system . A cell density of 1 × 105 cells  were seeded on round cover slips (12 mm diameter) and were treated with 2,5 μg/ml 5A1 for 24 h, 48 h, 72 h and 96 h, respectively. For calcein-AM / PI double staining, cells were incubated in 1 μM calcein-AM  plus 1 μM PI  for 30 min at 37°C in the dark. Subsequently, cells were fixed with 4% paraformaldehyde for 15 min before placing cover slips upside down on an object slide using mounting medium . Fluorescence signals were analysed with Leica DM500B .Cell viability staining was performed by calcein-AM / PI double staining. A cell density of 1 × 10Migration assays were performed as previously described . Migrati5 MCF-7 or 410.4 breast cancer cells were seeded into the upper well of the chamber in 500 μl medium, the lower well was filled with 1 ml of the same medium. For co-culture experiments 1,5 × 105 MCs or 2 × 105 MG were seeded in transwell inserts  in 500 μl medium. The medium used was either DMEM + 10% FCS (co-cultures with MG) or DMEM + 10% FCS + 15% L929 conditioned medium (co-cultures with MCs). The transwells were inserted into the upper well of the Boyden chamber and anti-CSF-1 was added. After 96 h the floating and adherent carcinoma cells in the lower well were removed and counted. All experiments were performed at least in triplicate.Invasion was measured in a modified Boyden chamber as described previously . BrieflyRNA from tissue was isolated with a modified Trizol  method incorporating a DNaseI  digestion step. RNA from cells was isolated using the “High Pure RNA isolation kit” . Reverse transcription was performed with the iScript Master Mix .Quantitative RT-PCR was performed as previously described . The folCells were lysed and homogenized in RIPA lysis buffer . Up to 30-60 μg of total protein were subjected to SDS-PAGE (10%) and blotted onto a nitrocellulose membrane (Amersham Biosciences). Ponceau S staining was used as loading control. Membranes were incubated with antibodies specific to AKT , pAKT , S6 , pS6  and anti-human-CSF-1 . Signals were detected with ECL Prime (Amersham Biosciences).t-test the significance of the differences between groups in the qRT-PCR, Boyden chamber, MTT experiments etc. was tested and p-values < 0.05 were considered significant.Using the Student's genefu r-package [pam50 intrinsic genes list option for subtype prediction [http://www.r-project.org).Three public microarray datasets were retrieved from NCBI Gene Expression Omnibus (GEO)  data rep-package , 38 withediction . All ana


Benzodiazepines are used in many of settings to induce sedation, but can cause a reduction in respiratory drive. Objective monitoring of the effect of benzodiazepines on respiratory status in non-intubated patients has been difficult, putting patient safety at risk. A non-invasive respiratory volume monitor (RVM) that provides continuous measurement of minute ventilation (MV), tidal volume (TV) and respiratory rate (RR) was used to quantify the effects of midazolam on respiratory status in spontaneously breathing patients.An impedance-based RVM  was used in 30 patients who received 2 mg midazolam prior to induction of anesthesia and were sedated but spontaneously breathing. Eleven of these patients  received midazolam at least 20 minutes prior to induction. Digital RVM data were collected and MV, TV and RR calculated and evaluated from 30-second segments 10 minutes before and after the first dose of midazolam. Ten patients were analyzed as a group and one patient was analyzed separately (due to idiosyncratic reaction).P < 0.01, both) while RR remained essentially unchanged . In the younger half of the cohort (45 ± 16 years), the decreases in MV and TV were not significant, only 6 ± 3% and 8 ± 5%, respectively. The older half of the cohort (72 ± 8 years) displayed fourfold greater MV and TV decreases , when compared with the younger cohort, P < 0.01, Figure Following administration of midazolam, the group MV and TV decreased an average of 19 ± 7% and 16 ± 5%, respectively (mean ± SEM, Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications.


Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological functions. The possible roles of oxysterols in various neurological diseases makes the analysis of these metabolites in the central nervous system of particular interest. Here, we report the identification and quantification of a panel of twelve sterols in mouse cerebrospinal fluid (CSF) using liquid chromatography–mass spectrometry exploiting enzyme assisted derivatisation for sterol analysis technology. We found low levels of oxysterols and cholestenoic acids in CSF in the range of 5 pg/mL–2.6 ng/mL. As found in man, these concentrations are one to two orders of magnitude lower than in plasma. Oxidation of cholesterol is catalysed by numerous sterol hydroxylases, primarily members of the cytochrome P450 (CYP) family of enzymes vitro and in vivoThe high level of cholesterol in the brain (approximately 2% wet weight of brain) has stimulated research investigating altered cholesterol metabolism as a causative factor in neurological diseases. For example, oxysterol levels have been shown to be perturbed in neurodegenerative diseases including Alzheimer’s disease Mouse models of neurological diseases are an important resource to investigate the causes and treatments of these conditions. Increasingly sophisticated mouse models for numerous diseases are becoming available, as well as knockout animals with mutations of specific enzymes involved in sterol homeostasis. To link observed biological phenomena to the underlying molecular causes it is useful to analyse the levels of oxysterols and cholestenoic acids in different tissues of the mouse. Blood plasma is often used for analysis as it is straightforward to collect and gives a “snapshot” of oxysterol levels throughout the body. However, the presence of the blood brain barrier (BBB) raises questions of the relevance of plasma measurements in the context of neurological diseases. Previously we have analysed brain tissue which circumvents this problem and allows direct analysis of metabolites that may play a role in disease An alternative is to measure oxysterols in cerebrospinal fluid (CSF). This has the advantage of relatively straightforward sample collection in humans while representing conditions in the central nervous system (CNS) more faithfully than blood plasma. We have previously reported the identification and quantitation of about 25 oxysterols and cholestenoic acids in human CSF Here, we present a robust method for the identification and quantification of cholesterol metabolites in mouse CSF that opens new possibilities for biomarker discovery and investigations into the underlying causes of neurological diseases. We compare this data with concentrations of oxysterols and cholestenoic acids found in plasma to give an overview of cholesterol metabolism in mouse.22.1CSF was from male (∼40 g) and female (∼34 g) Swiss/Webster mice (4–6 months), sedated with an overdose of Nembutal (150 μg/g body weight). CSF was collected from the cisterna magna as described by Liu and Duff 2.2n) by the method previously described Oxysterols and cholestenoic acids from mouse plasma were analysed using enzyme assisted derivatisation for sterol analysis (EADSA) and liquid chromatography–tandem mass spectrometry (LC–MS2.32H7]hydroxycholesterol ([2H7]24R/S-HC) (2 ng), 22R-hydroxycholest-4-en-3-one ([2H7]22R-HCO) (2 ng), 7α,25-dihydroxycholesterol  (0.2 ng) and cholesterol (20 μg)  in an ultrasound bath. After 5 min water (Fisher Scientific) was added to give a final volume of 1.5 mL of 70% ethanol. The mixture was centrifuged for 30 min at 17,089×g at 4 °C. To remove cholesterol from the sample a solid phase extraction (SPE) step was used. A 200 mg tC18 Sep-Pak cartridge (Waters) was preconditioned with ethanol (4 mL) followed by 70% ethanol (6 mL) after which the sample was loaded. The flow-through was combined with a wash of 70% ethanol (5.5 mL) to give a 7 mL fraction containing oxysterols and cholestenoic acids. The solvent was evaporated in a vacuum centrifuge overnight.CSF (54–100 μL) was added to ethanol  containing 24R/S-+ fragment in MS2 corresponding to the loss of pyridine, the [M-79]+ ion was isolated and when activated further gave structurally informative MS3 spectra.Oxysterols were separated using a RSLC nano Ultimate 3000 (Dionex) with a Hypersil Gold C33.1We used EADSA to charge-tag sterols of interest with the GP reagent . EnzymatTo account for the very low levels of oxysterols (pg/mL) in mouse CSF, as well as the small amount of fluid available , we used pooled CSF samples (6–8 mice) and made several modifications to our procedure usually employed for plasma analysis. We used a smaller SPE cartridge (50 mg of sorbent cf. 200 mg for plasma) for the removal of the excess derivatisation reagent. In addition, before analysis we concentrated samples in a vacuum centrifuge to maximize the amount of analyte injected on the LC column. We worked up 100 μL of CSF pooled from male mice, and 54 μL of CSF pooled from females and injected 85% of each in a single injection.m/z error of <5 ppm. By plotting reconstructed ion chromatograms (RICs) for m/z values corresponding to different oxysterols, we could detect multiple metabolites. For example, a RIC for m/z 534.4054 shows the side-chain monohydroxycholesterols 24S-HC, 25-HC and 26-HC along with the B-ring oxidised metabolites 7α-HC, 7β-HC, 7-oxocholesterol (7-OC) and 6-HC  plus 7α-hydroxy-3-oxocholest-4-enoic acid   . Note, uH,3O-CA)  and ster in vivo . In addi in vivo .n spectra recorded in the LIT of the LTQ-Orbitrap. Fragmentation of sterols tagged with the GP reagent gives a characteristic peak at [M-79]+ corresponding to the loss of pyridine (3) then gives structurally informative fragments unique to each individual metabolite. For example, 3 spectrum of 24S-HC with fragments characteristic of a GP derivatised sterol, without a 7-hydroxy group present, at m/z 151.1, 163.1 and 177.1 along with compound specific fragments such as the distinctive peak at m/z 353.4. The major peak at m/z 437 corresponds to [M-79-H2O]. Other oxysterols and cholestenoic acids give different fragmentation patterns that can be compared with authentic standards to confirm the identity of each metabolite