Over the past 16 years, sepsis management has been guided by large-volume fluid administration to achieve certain hemodynamic optimization as advocated in the Rivers protocol. However, the safety of such practice has been questioned because large-volume fluid administration is associated with fluid overload and carries the worst outcome in patients with sepsis. Researchers in multiple studies have declared that using less fluid leads to increased survival, but they did not describe how to administer fluids in a timely and appropriate manner.An 86-year-old previously healthy Sundanese man was admitted to the intensive care unit at our institution with septic shock, acute kidney injury, and respiratory distress. Standard care was implemented during his initial care in the high-care unit; nevertheless, his condition worsened, and he was transferred to the intensive care unit. We describe the timing of fluid administration and elaborate on the amount of fluids needed using a conservative fluid regimen in a continuum of resuscitated sepsis.Because fluid depletion in septic shock is caused by capillary leak and pathologic vasoplegia, continuation of fluid administration will drive intravascular fluid into the interstitial space, thereby producing marked tissue edema and disrupting vital oxygenation. Thus, fluids have the power to heal or kill. Therefore, management of patients with sepsis should entail early vasopressors with adequate fluid resuscitation followed by a conservative fluid regimen. AlthougAdministration of intravenous fluid remains one of the most common therapies given to hospitalized patients; however, studies have shown that up to 20% of these patients are given inappropriate fluid therapy . As subtet al. divided fluid administration into four phases: resuscitation, optimization, stabilization, and evacuation (ROSE) [With new insights into fluid administration and clinical outcome, perhaps the use of large-volume fluid resuscitation in the management of patients with sepsis ought to be reconsidered. How much fluid is needed in what amount of time, and what are the parameters for monitoring a safe and adequate fluid balance? In a review on intravenous fluid therapy, Hoste n (ROSE) . In thisA previously healthy 86-year-old Sundanese man with no comorbidities was admitted to the general ward of our hospital with excruciating pain in his right hip and knee after a prior fall. Our patient weighed 65 kg and was 165 cm tall. On admission, he was fully alert, and the results of his radiologic investigations were normal. Intravenous analgesics and nerve blocks were administered, and the patient remained hospitalized for 12 days of nursing care. On the 12th day, intravenous catheter site induration and redness developed, which rapidly progressed to necrotic and pustular tissue formation within 12 h , platelets (Plt) 137,700/μl , C-reactive protein 199.90 mg/L (reference range 0.00–3.00 mg/L), and procalcitonin (PCT) 37.00 ng/ml (reference range <0.5 ng/ml). Other levels recorded were alanine aminotransferase 13 U/L (reference range 0–55 U/L), aspartate transaminase 12 U/L (reference range 5–34 U/L), urea 126.0 mg/dl (reference range <50 mg/dl), creatinine 2.42 mg/dl (reference range 0.5–1.1 mg/dl), Na+ 131 mEq/L (reference range 135–145 mEq/L), K+ 6.2 mEq/L (reference range 3.5–5 mEq/L), Cl− 101 mEq/L (reference range 96–110 mEq/L), and random blood glucose 114 mg/dl.Our patient was moved to the high-care unit (HCU) with the following hemodynamic parameters: blood pressure 107/46 mmHg, mean arterial pressure (MAP) 66 mmHg, heart rate 88 beats per minute (bpm), respiratory rate 21 breaths per minute, and oxygen saturation 100% through a 5-L nasal cannula. He remained afebrile. A complete blood examination revealed hemoglobin (Hgb) 1.94 g/dl (reference range 11.70–15.50 g/dl), hematocrit (Hct) 32.35% (reference range 35.00–47.00%), white blood cell count (WBC) 20,550/mmA diagnosis of necrotizing fasciitis with sepsis, stage 2 acute kidney injury, and hyperkalemia was made. One gram of intravenous cefoperazone twice daily and 400 mg of moxifloxacin once daily were given. The patient’s hyperkalemia was treated using 25 U of insulin and 100 ml of 40% dextrose solution for 2 h. A nasogastric tube was inserted, and the patient’s stomach was decompressed. A central venous catheter was inserted, and cultures from blood, urine, and sputum were taken.2) 77.9 mmHg, partial pressure of oxygen (pO2) 94 mmHg, HCO3− 20.9 mEq/L, base excess −10 mEq/L, and serum lactate 3.3 mmol/L (reference range <0.6–2.2 mmol/L). The patient’s blood pressure continued to fall and reached 60/30 mmHg (MAP 40 mmHg), followed by multiple episodes of bradycardia from 140 bpm to 70 bpm despite administration of 500 ml of colloid and 100 ml of 20% albumin. Hence, noradrenaline at 0.5 μg/kg/minute and dobutamine at 10 μg/kg/minute were initiated. In the HCU, the patient received a total fluid input of 4644 ml with urine output of 55 ml/h and fluid balance of +3540 ml/20 h.Nevertheless, the patient’s condition worsened. He became unresponsive with a respiratory rate of 38 breaths per minute and prominent use of accessory muscles. His oxygen saturation was 88% with a 15-L non-rebreathing mask; his central venous pressure (CVP) was 5 mmHg; his blood pressure was 90/60 mmHg (MAP 70 mmHg); and he had an electrocardiographic reading of atrial fibrillation with rapid ventricular response and a heart rate of 140–160 bpm. Arterial blood gas analysis revealed respiratory acidosis with pH 7.029, partial pressure of carbon dioxide , and his CVP was 16 mmHg. Noradrenaline was increased to 0.8 μg/kg/minute and dobutamine to 3 μg/kg/minute, to which he responded. His blood pressure was maintained at 115/60 mmHg (MAP 78 mmHg); his heart rate was 110–120 bpm; and his CVP was 12 mmHg. Two hours postintubation, his blood gas analysis revealed pH 7.28, pCO2 39.6 mmHg, pO2 112.5 mmHg, HCO3− 19.1 mEq/L, base excess −6.9 mEq/L, and a lactate level decreasing to 2.27 mmol/L. A chest x-ray revealed patchy infiltrates on the lower lung regions with a cardiothoracic ratio of 61% , where he was intubated and mechanically ventilated. He was placed on adaptive support ventilation mode with a positive end-expiratory pressure of 5 cmHContinuous analgesia and sedation with morphine and midazolam infusion were administered, and the patient’s vital signs stabilized. A repeat blood workup revealed insignificant changes except for urea and creatinine increasing to 159.5 mg/dl and 2.74 mg/dl, respectively. The patient’s PCT levels spiked to 97.60 ng/ml, and antibiotics were switched to meropenem 1 g every 8 h, moxifloxacin 400 mg once daily, and 200 mg fluconazole twice daily. At that time, our patient received 1000 kcal/500 ml of parenteral nutrition through intermittent bolus nasogastric feeding tubes.Streptococcus pyogenes, and meropenem was changed to 400 mg of teicoplanin daily along with moxifloxacin, based upon antibiotic sensitivity results. Cultures from the patient’s blood and urine revealed no growth, whereas a sputum culture revealed growth of Candida albicans, and a fluconazole regimen was resumed. The patient’s blood gas analysis normalized with pH 7.38, pCO2 40.6 mmHg, pO2 138.8 mmHg, HCO3− 16.6 mEq/L, base excess −3.9 mEq/L, and serum lactate 1.3 mmol/L. His blood pressure was stable at 110/50 mmHg (MAP 70 mmHg); his heart rate was 100–120 bpm with atrial fibrillation; and his CVP was 12 mmHg. Intravenous amiodarone at 150 mg for 10 minutes was given, followed by a continuous infusion of 150 mg for 12 h.On the second day, the results of a wound culture revealed the growth of Wound debridement and necrotomy were performed on the second day. However, 1 h postdebridement, the patient’s blood pressure plummeted to 50/30 mmHg (MAP 38) with a heart rate of 100 bpm. A bolus of 100 ml of normal saline was given along with noradrenaline at 0.8 μg/kg/minute and epinephrine at 8 μg/kg/minute. The amiodarone infusion was stopped. The patient’s vital signs responded progressively, and epinephrine was slowly tapered and then completely discontinued after 2 h. Maintenance fluids were given at 40 ml/h normal saline with a total daily fluid input of 3850 ml, diuresis of 70 ml/h, and a daily fluid balance of +1255 ml.3 , and a PCT level decreasing to 83.46 ng/ml. His coagulation profile revealed Plt 149,000/μl  with a prothrombin time (PT) of 13.60 seconds, international normalized ratio (INR) of 1.15, activated partial thromboplastin time (aPTT) of 55.40 seconds, and D-dimer of 5.36 ng/ml. His urea decreased slightly to 151.7 mg/dl (reference range <50 mg/dl); his creatinine was 1.84 mg/dl; and his serum albumin was 2.88 mg/dl (reference range 3.5–5.3 mg/dl). Enteral nutrition was resumed because no residual gastric fluid was noted, and the maintenance fluid used was normal saline at 20 ml/h with noradrenaline tapered to 0.01 μg/kg/minute. Total daily fluid input was 2198 ml with diuresis of 91 ml/h and a fluid balance of −967 ml.On the third day, the patient’s mental status improved dramatically; he was able to respond to instructions, and his vital signs remained within normal limits. The ventilator mode and settings remained unchanged, and the patient was actively triggering breaths with good ventilator synchrony. A complete blood examination revealed Hgb 9.9 g/dl (reference range 11.70–15.50 g/dl), Hct 24.5% (reference range 35.00–47.00%), WBC 24,190/mmOn the fourth day, the vasopressor infusion was discontinued. The patient remained afebrile and responsive; hence, weaning from mechanical ventilation was initiated. His vital signs remained stable throughout the weaning process, with a blood pressure of 110/70 mmHg (MAP 83 mmHg), heart rate of 85–90 bpm, and CVP of 9 mmHg. His physical examination revealed clear lung sounds confirmed by a clear chest x-ray, and the results of his arterial blood gas analysis were within normal limits. The maintenance fluid used was normal saline at 40 ml/h with a total daily fluid input of 2610 ml, diuresis of 100 ml/h, and a fluid balance of −765 ml.2 26.4 mmHg, pO2 173.1 mmHg, HCO3− −17.8 mEq/L, and base excess −5.1 mEq/L. A repeat blood workup revealed Hgb 10.28 g/dl, Hct 31%, WBC 17,380/mm3, and Plt 114,000/μl. Other readings were PT 14.60 seconds, INR 1.24, aPTT 43.80 seconds, and D-dimer 5.90. The patient’s urea level was 130.9 mg/dl, and his creatinine level was 1.24 mg/dl. His nasogastric tube was withdrawn, and he was started on oral feeding. Normal saline was given at 20 ml/h with a total daily fluid input of 2562 ml, diuresis of 148 ml/h, and a daily fluid balance of −1998 ml . Fluid output was counted as fluids collected from urine, wound drainage, nasogastric fluids, and insensible water loss, which was calculated at 15% of body weight in milliliters (975 ml/day) Fig. .Fig. 4DaNecrotizing fasciitis is a rapid and progressive necrotizing process involving the subcutaneous fat, superficial fascia, and superficial deep fascia . The diaAs the patient’s sepsis progressed, he experienced respiratory distress, which may have been a result of leaky capillaries at the arterial-alveolar junction. Edema on alveolar cells changes their vital cell architecture because less surface area is available for effective gas exchange . ImpaireThroughout his septic period, the patient experienced an episode of hypotension that was unresponsive to fluid administration. With a massive “cytokine storm,” profound vasodilation and capillary leak occur, resulting in rapid fluid distribution into the interstitial space, thus leaving the intravascular space devoid of effective circulating volume and creating hypotension , 8. TherIn multiple studies of intravenous fluid resuscitation using either early goal-directed therapy or standard care, researchers have reported an average of more than 4 L of fluid administered during the initial 6 h of resuscitation –12, wherAt this point, the clinician needs to be wary in instituting further fluids because doing so will increase edema, especially to organs such as the liver and kidney. With progressive capillary leak, these encapsulated organs are unable to compensate for the increased volume, and severe interstitial edema will compress vital blood flow . Aside fet al. best summed up the four stages of fluid therapy as divided into resuscitation, optimization, stabilization, and evacuation phases [We believe fluid therapy is best tailored to specific indications and that the administration of aggressive fluid administration should be restricted only to the resuscitation phase of septic shock. Hoste n phases . Our patAfter the patient’s shock was managed, he entered the optimization phase, which is a state of compensated shock. At this phase, giving too little fluid will cause the patient to fall back into a shocked state, whereas giving too much fluid will cause inadvertent fluid overload . ConsequNext comes the stabilization phase, which is the period when fluids are administered to provide daily requirements and replace ongoing losses, if any . The FluIn the evacuation phase of fluid therapy, fluids are deliberately removed from the patient, and the goal is to strive for a negative fluid balance . In our Streptococcus during surgery spark an immunological response followed by massive release of histamine into the circulation, and they produce marked hypotension [Candida albicans.Source control through wound debridement and necrotomy was performed, and 1 h postdebridement our patient experienced sudden hypotension that may have been due to toxic shock syndrome. Toxins liberated from otension . A bolusAnother variable commonly sought in the management of patients with sepsis is the CVP. Throughout our patient’s care, his CVP value was not used as a guide in titrating fluid administration, because this method has been proven to be an unreliable method of measuring overall volume status. The heart and systemic vasculature is a complex system that functions together in preserving effective circulating volume by mobilizing fluids through vasoconstriction, vasodilation, or increasing cardiac output . The resAside from fluid input, an important aspect of improving clinical outcomes of patients with sepsis is to keep a watchful eye on their fluid balance. Cumulative fluid balance reported by critically ill nonsurvivors averaged 7761 ± 7391.9 ml for 1 week . Our patThroughout his ICU stay, our patient received normal saline and additional fluids from enteral nutrition at 1000 kcal/500 ml, which were given as intermittent 100-ml bolus feedings. Deep vein thrombosis prophylaxis was achieved through mechanical compression because thrombocytopenia, coagulopathy, and elevated D-dimer levels herald the development of disseminated intravascular coagulation and heparin was of limited use. Passive physiotherapy was initiated early to prevent muscle atrophy and stiffness, whereas active physiotherapy followed after the patient responded . BronchiCurrent evidence on fluids and sepsis urges us to reconsider the fluid regimen in the management of patients with sepsis because aggressive fluid administration after a state of resuscitated sepsis is well-documented to have the worst outcome. Patients with sepsis respond poorly to fluids because a massive and erratic cytokine storm results in arteriovenodilation and microcirculatory dysfunction during the early stages of septic shock; hence, fluid administered during the resuscitation phase is best given with vasopressors and early. After this phase, fluids must be tapered to prevent inadvertent fluid overload, which will worsen oxygen transport at the cellular level. Successful management of sepsis requires an integrated approach of infection control, use of appropriate antimicrobials, and supportive care. But perhaps every clinician ought to be extra vigilant in prescribing the most routine drug of all, fluids.


The former and latter sealing timescales correspond to the extensional stress period (~3 × 101 years) and the recurrence interval of megaearthquakes in the Nankai Trough (~3 × 102 years), respectively. We therefore suggest that silica precipitation in the accretionary wedge controls the recurrence interval of large earthquakes in subduction zones.Silica precipitation is assumed to play a significant role in post-earthquake recovery of the mechanical and hydrological properties of seismogenic zones. However, the relationship between the widespread quartz veins around seismogenic zones and earthquake recurrence is poorly understood. Here we propose a novel model of quartz vein formation associated with fluid advection from host rocks and silica precipitation in a crack, in order to quantify the timescale of crack sealing. When applied to sets of extensional quartz veins around the Nobeoka Thrust of SW Japan, an ancient seismogenic splay fault, our model indicates that a fluid pressure drop of 10–25 MPa facilitates the formation of typical extensional quartz veins over a period of 6.6 × 10 However, many unresolved factors add ambiguity to any discussion of the timescales of seismic cycles, including which parameters control earthquake triggering and the timescales required to change crustal conditions from aseismic to seismic. It has been suggested that the distribution of high pore pressures within the seismogenic zone controls earthquake occurrence, as inferred from a combination of seismic data, the porosity of in situ rocks, and numerical simulations7. The interseismic period related to the cycling of fluid pressure has been explained by changes in tectonic stress and fault strength8. In particular, the buildup and release of pore fluid pressure during a seismic cycle promote temporal changes in fault strength. The dissolution and precipitation of minerals play an important role in closing dilatational fractures generated during the co-seismic period and reducing the porosity of rocks9. However, the evolution of relevant hydrological properties  related to water–rock interaction during the interseismic period is poorly understood.Earthquake recurrence intervals are commonly used to predict the timescales of future earthquakes12. Silica–water interaction may play a crucial role in a wide range of seismic phenomena; e.g., the rapid precipitation of large amounts of silica due to a large pressure drop in response to an earthquake13, fault weakening by silica gel formation on fault surfaces15, and pressure solution and diffusion of silica that occur in relation to slow slip earthquakes16. In particular, silica precipitation effectively seals cracks and faults, and could therefore significantly influence the buildup of fluid pressure during the interseismic period18. However, no studies have quantitatively discussed the timescales of quartz vein formation and earthquake recurrence intervals within subduction zones.Silica is a dominant component of crustal rocks, and widespread quartz veins in subduction zones at seismogenic depths are taken as proof of significant fluid flow and silica precipitationHere we propose a novel kinetic model of quartz vein formation that allows us to estimate the sealing times of isolated cracks based on pore-fluid pressure drop within the crack, advective flow of silica-rich fluids from the peripheral host rock, and quartz crystal growth in the seismic zone. Our model quantifies the relationship between the recurrence intervals of earthquakes and the sealing timescales of quartz-filled extensional cracks around the Nobeoka Thrust, southeastern Japan, a typical seismogenic megasplay fault in an ancient subduction zone.19  axis being almost horizontal and trending roughly NNW−SSE in both the hanging wall and footwall21 24, indicates that the normal faulting type stress regime at the time of fracture opening was secondary stress generated by slip of the Nobeoka Thrust21. The temporal stress changes are similar to those of Mw 9.0 2011 Tohoku-Oki earthquake25 and to large trench-type earthquakes along the décollement26. The microstructural features of syntectonic mineral veins have been interpreted as evidence of temporal fluctuations in fluid pressure during repeated earthquake cycles within a seismogenic megasplay fault in an ancient subduction zone22. The extensional veins are consistent with multiple episodes of slip on the Nobeoka Thrust. Analyses of fluid inclusions hosted by rocks of the Shimanto belt in Kyushu reveal that the fluid salinity is similar to or lower than that of seawater27. Geochemical and mineralogical analyses suggest that extensional veins around the Nobeoka Thrust formed from relatively oxidized, locally derived pore fluids with neutral pH22. Analyses of vitrinite reflectance and fluid inclusions reveal that vein formation occurs under both high P–T conditions  and low P–T conditions (140–250 °C and 150–190 MPa) in the hanging wall and footwall of the Nobeoka Thrust29. The difference in temperature across the fault was generated by activation of the Nobeoka Thrust as an out-of-sequence-thrust or megasplay fault19. The normalized pore fluid pressure ratio, the lower bound of the maximum fluid pressure level30, is ~0.95 for a tensile strength of 10 MPa21. This stress condition means that the tensile overpressure exceeds σ3 during the period of vein formation. The formation of high-angle extensional fractures occurs only when tensile overpressure Pf > σ3 is attained31.The veins around the Nobeoka Thrust are classified into three types: extensional crack-filling veins (mode I cracks), fault-filling veins, and post-mélange veinsl21 Fig. . The neg1–3 × 102 years in the period covered by historical records (684–1946 A.D.)2. The high pore pressures were expected to be close to lithostatic pressure along the mega-splay fault of the Nankai Through7. However, there is no consensus on the recurrence intervals of earthquakes on splay/megasplay faults around the Nobeoka Thrust or those of megathrust earthquakes along the Nankai Trough. If we treat the postseismic period (i.e. the period under the secondary stress regime) as having effectively zero aftershock activity after roughly one-tenth of the recurrence interval , the timescale required to open extensional cracks in the Shimanto accretionary wedge is 9 × 100–3 × 101 years.Great earthquakes along the Nankai Trough occurred at intervals of 9 × 10wv) and lengths (lv) of quartz veins were estimated from eight thin sections (48 × 22 mm) and field measurements, respectively; both measurements are log-normally distributed, with wv = 1.3 × 101–3.4 × 102 μm , with a few veins being longer than 5.0 × 101 cm .We observed discrete extensional veins in the hanging wall of the Nobeoka Thrust, filled mainly with quartz and lesser calcite. The aperture widths  induces fluid advection from the host rock into the crack and silica precipitation in the crack. The two sides of the circular crack walls are the input paths of fluid from the host rock into the crack, whereas fluid flows radially through the crack for a distance equal to the radius. Based on the quartz vein textures  the fluid volume required for sufficient silica precipitation to seal a crack completely, which depends on the pressure drop and the kinetics of silica precipitation and (2) the fluid volume of the reservoir that supplies SiO2-supersaturated fluid by advection, which follows Darcy’s law. We solve for the unique sealing time (ts) simultaneously by iterating with the constraint that the two fluid volumes are equal (Supplementary Method).We propose a model of quartz vein formation associated with fluid advection from host rocks and silica precipitation in a crack (Supplementary Method). An isolated disk-shaped crack exists at the center of a spherical host-rock reservoir of radius ers Fig. . Pore fles Figs. , we cons22 and low salinity27, and we assume a constant SiO2 concentration in the fluid in the host rock (CSiO2 = 6.0 × 102 mg/kg(H2O)), which is taken from the solubility of quartz in pure water at 250 °C and 260 MPa  is 6.0 × 102 mg/kg(H2O) as a result of pure water saturated via quartz at 250 °C and 260 MPa , which are the geometric mean values of each parameter for extensional veins along the Nobeoka Thrust 35. The porosity of the host rock ranges from 3% to 10%, as measured by geophysical wireline logs across the Nobeoka Thrust23. In this study, we use a minimum porosity of 3% for most of the calculations, and we show that porosity has a minor influence on estimates of sealing time. The porosity and permeability of the host rock are set to be constant during crack sealing because the volume of quartz required to seal a crack is much smaller than that of the fluid reservoir in the host rock.In examining the formation of extensional cracks, we consider the realistic conditions around the Nobeoka Thrust (Supplementary Note). The initial pore fluid pressure and temperature of the extensional crack are set to 260 MPa and 250 °C, respectively, which are equivalent to the lithostatic conditions at a depth of 10 km on the Nobeoka Thrust Fig. . The veiMPa Fig. 34. The Sst Figs. . We use P from 0.1 to 160 MPa, so that the pore fluid pressure in the crack decreases from lithostatic to hydrostatic  increases from 7.1 × 10−5 to 1.9 × 10−1, and the time taken for complete sealing of a crack becomes much shorter: for a typical crack, with wv = 5.2 × 101 μm and lv = 7.4 × 100 cm, the time decreases from 3.1 × 106 years (3.1 My) to 7.3 × 10−2 years (27 days)  decreases to a level close to the solubility of quartz at each Pcrack. Accordingly, the difference in SiO2 concentration between the input and output fluid, which corresponds to the amount of silica precipitation, increases from 4.2 × 10−2 mg/kg(H2O) at ΔP = 0.1 MPa to 9.4 × 101 mg/kg(H2O) at ΔP = 160 MPa  at T = 250 °C, the sealing time (ty) in years can be expressed as a logarithmic–linear function:lv = 7.4 × 10–1 cm) becomes similar to that of a mean-length crack with increasing ΔP  Fig. , where t1 m Fig. . While pMPa Fig. . In this ΔP Fig. . In a sm ΔP Fig. .Figure 4P < ~25 MPa) during an earthquake36. In addition, assuming a rock tensile strength of 10 MPa, cracks remain open in the range ΔP = 0–10 MPa. Hence, an appropriate range of ΔP for the formation of extensional quartz veins at around the Nobeoka Thrust is 10–25 MPa, for which the sealing time of a geometric mean size crack is 6.6 × 100–5.6 × 101 years  ranges from 1.4 × 100 to 1.2 × 101 years at ΔP = 10–25 MPa , the sealing time of a geometric mean size crack is 4.4 × 10–1–3.8 × 100 years, while in the case of lower porosity (3%) and permeability (1 × 10–20 m2) the sealing time is between 6.6 × 101 years and 5.6 × 102 years at ΔP = 10–25 MPa , fluid pressure generated by extensional cracks can drop by up to 10% of lithostatic pressure . In the possible range of fluid pressure drops along the Nobeoka Thrust (ΔP = 10–25 MPa), the sealing time in the model of this study is 101–102 times faster than in the diffusive model  by 225 all possible combinations of aperture width  determined by the presence probability of each volume of veins with a cumulative number index of sealed crack (ωt = 0–1), which is a rate between a cumulative number of completely sealed crack and total number of possible length-aperture width combinations obtained in this study (Supplementary Method). We assume that all cracks are opened  at the time of earthquake rupture (t = 0) to show the range of duration of the porosity recovery period, and then Ωt will decrease toward zero and ωt will increase toward one by silica precipitation, respectively (Supplementary Method). This calculation does not consider the porosity of the host rock. Figure P = 25 MPa could recover by 89% (Ωt = 0.1) to >99% (Ωt < 0.01) within 9 × 101–3 × 102 years, respectively. The cumulative frequency of complete sealed crack number is 89% within 90 years  to 66% (Ωt = 0.34) within 9 × 101–3 × 102 years, respectively  within 8 × 102 years. The cumulative frequency of complete sealed crack number is 45% within 9 × 101 years, which is almost half of that at ΔP = 25 MPa  inferred for the extensional quartz veins around the Nobeoka Thrust indicate that advection was the dominant transport mechanism during quartz vein formation.The sealing time of a crack depends on various factors, including porosity and permeability in the host rock, and fluid composition. For the area around the Nobeoka Thrust, estimates of sealing time are more strongly influenced by the permeability of the host rocks than the porosity. An increase in host-rock porosity from 3% to 10% reduces the sealing time by ~30% Fig. , and an ars Fig. . Several ΔP Fig. . The ΔP ity Fig. . Accordi34 and the rate of quartz dissolution–precipitation38. For example, at ΔP = ~160 MPa, quartz solubility in seawater is ~1.6 × 101 mg/kg(H2O) lower than in pure water34 (Supplementary Note) and the rate of quartz dissolution in seawater is about one order higher than that in pure water38. Therefore, the sealing time of extensional quartz veins that formed from fluids with compositions similar to those that contributed to vein formation around the Nobeoka Thrust  could be slightly shorter than that estimated in this study based on an assumption of pure water.The salinity and pH of a fluid affect quartz solubility–7 m2). One system comprises a single crack  and the other a cluster of 100 thin cracks of the same size . At ΔP = 25 MPa, the sealing time of the cluster (2.1 × 100 years) is ~102 times faster than that of the single crack (2.1 × 102 years) because of the larger surface area for quartz precipitation 2. For complete sealing of larger cracks, silica precipitation should continue in the openings of larger cracks even after the stress regime changes to compression, because our field studies revealed no evidence for multiple crack–seal events in any single extensional quartz vein (Fig. 49; thus, collapse of the cracks is prevented, even after a change in stress regime.The recurrence intervals of subduction thrust earthquakesein Fig. . Conside2 years (Fig. P = 25 MPa could recover by >99% within ~3 × 102 years (Fig. 2. This result suggests that when megaearthquakes form extensional cracks in the accretionary wedge, the porosity is nearly recovered by silica precipitation within the aseismic interval, regardless of crack size. Figure –1–1.0 × 100 m, as required to seal a crack of mean size in the model of this study (Fig. Under the conditions expected along the Nobeoka Thrust, almost all extensional quartz veins form within ~1 × 10ars Fig. , and thears Fig.  known reudy Fig. , is greaudy Fig. , our mod50, which may result in increased fluid pressure due to sealing of flow paths and supplying of fluid from deeper parts22, thereby leading to an increased probability of earthquake occurrence51. The fault-valve model8 describes a cycle wherein instantaneous permeability rises and fluid pressure drops at the time of an earthquake; these quantities then decrease and rise again, respectively, as the fault approaches the next earthquake. Therefore, the geochemical evolution of accretionary wedge porosity, as proposed in this study, relates to the evolution of permeability and fluid pressure in the earthquake cycle. Decreasing porosity enhances the build-up of pore fluid pressure because the fluid is supplied by flowing or percolating in the bulk and along faults or décollement during both pre- and post-seismic27. Pore pressure builds-up with increasing the number of closed extension cracks, which may not be in one-to-one relationship, and its timescale is similar to that of crack sealing. The high porosity generated at the time of an earthquake under extensional stress is followed by a rapid decrease just after the earthquake. During the low porosity regime, when the likelihood of the next earthquake is already high, porosity still decrease due to silica precipitation in larger cracks when the pressure difference between the host rock and cracks is maintained before an earthquake (Fig. A decrease in sandstone porosity leads to the permeability decrease as recognized in various hydrostatic compression testsake Fig. . We concThe datasets generated during the current study are available from the corresponding author on reasonable request.Supplementary information


We investigate the relationship between social media, Twitter in particular, and stock market. We provide an in-depth analysis of the Twitter volume and sentiment about the 30 companies in the Dow Jones Industrial Average index, over a period of three years. We focus on Earnings Announcements and show that there is a considerable difference with respect to when the announcements are made: before the market opens or after the market closes. The two different timings of the Earnings Announcements were already investigated in the financial literature, but not yet in the social media. We analyze the differences in terms of the Twitter volumes, cumulative abnormal returns, trade returns, and earnings surprises. We report mixed results. On the one hand, we show that the Twitter sentiment (the collective opinion of the users) on the day of the announcement very well reflects the stock moves on the same day. We demonstrate this by applying the event study methodology, where the polarity of the Earnings Announcements is computed from the Twitter sentiment. Cumulative abnormal returns are high (2–4%) and statistically significant. On the other hand, we find only weak predictive power of the Twitter sentiment one day in advance. It turns out that it is important how to account for the announcements made after the market closes. These after-hours announcements draw high Twitter activity immediately, but volume and price changes in trading are observed only on the next day. On the day before the announcements, the Twitter volume is low, and the sentiment has very weak predictive power. A useful lesson learned is the importance of the proper alignment between the announcements, trading and Twitter data. It is now accepted that financial markets are not governed solely by rational behavior of investors, as captured by the efficient market hypothesis. Their decisions are also influenced by their subjective beliefs and expectations, and by the information from the Internet. Online news and social media provide large amounts of data, from which potentially useful information can be extracted. We are interested in collective opinion and expectations of investors in relation to financial markets. We analyze social media data from the Twitter micro-blogging platform in terms of the attention to the most important events, and the collective expectations about the market moves.Twitter is becoming an increasingly popular platform used to monitor and forecast financial markets. The first to show a clear relation between the Twitter mood indicators and Dow Jones Industrial Average (DJIA) were Bollen et al. –3. In geThe range of the methodologies for analyzing the relations consists of the Granger causality , 3, 6, oSeveral papers analyze the relation between Twitter and stock market for the aggregated indices only, e.g., DJIA , 3 or S&We overcome this limitation by focusing on the most interesting time periods of an individual company, the quarterly Earnings Announcements (EA). It turns out that the volume of Twitter posts around most of the EAs is substantially higher and allows to draw statistically significant conclusions. This type of analysis is enabled by the “event study” methodology , 16, useThere have been applications of the event study methodology to Twitter data already. We are aware of three recent works which analyze the Twitter sentiment data in relation to the stock price movement. Sprenger et al. , 11 analThere are several improvements over our previous work . In thisThere are several works in the financial literature, dealing with EAs and their timings, related to our work. Berkman et al.  observe Doyle et al.  find thaSchroff et al.  analyze A recent work by Alostad et al.  is closeThis work makes several contributions to the analysis of relations between social media and stock market. First, we find significant relation between the Twitter sentiment and EA returns on the days of the announcements, with cumulative abnormal returns around 2–4%. Second, we observe important differences between different timings of the announcements. The announcements before the market opens show lower cumulative abnormal returns in comparison to the announcements after the market closes. Third, based on these results, we test a simple trading strategy with buy/hold/sell at the market close of the day prior to the EA. Fourth, we compare the Twitter sentiment to the earnings surprise, a measure frequently used in event studies.An important conclusion from these results is the requirement for a proper setup of event studies and other methods which investigate relations between Twitter and stock market. It is important to take into account the exact timing of the events with respect to the market trading hours. The daily aggregation of the Twitter data and its alignment with the market data have an impact on the perceived predictive power of Twitter. When analyzing the data at the daily resolution one needs to attribute the Twitter posts created after the market closes to the next trading day.https://dx.doi.org/10.6084/m9.figshare.4036269. The terminology and abbreviations used throughout the paper are in In this section we provide details about the data used in the study. The data are about the 30 companies in the DJIA index in the period of three years, from June 1, 2013, to June 3, 2016. The data consist of the Earnings Announcement (EA) events, the stock market data, and the Twitter data. All the data were collected according to the Terms of Use and Service of the source websites, and are available at ES is defined as:prep is the reported price of a share in the EA report, while pest is the expected price, as estimated by financial analysts. We collected the EA data from the http://www.zacks.com website. The missing values of the timings were filled from the information issued by the companies themselves.The data regarding the EAs contains the exact timings of the announcements, as well as the reported and expected price of a share. The earnings surprise is the difference between the reported and the expected earnings of a company. The earnings surprise http://www.google.com/finance. It consists of the daily trading volume and closing prices of the 30 DJIA companies and the DJIA index. From the data we calculate daily returns and longer term trading returns. The daily return Rd, used in the calculation of Cumulative Abnormal Returns (event study applications), is defined as:pd denotes the closing price of the stock on day d. Consistent with the original event study  are the abnormal, actual, and expected normal returns, respectively. The normal performance model used in this work is the market model: it assumes a linear relation between the overall market return and the return of the stock. More details are given in [An event study is based on the premise that in order to correctly capture the impact of an event, a measure of abnormal price return must be defined. This measure is the actual price return minus the normal return of the stock during the event window. For company given in .CAR) from time τ1 to τ2 is the sum of the abnormal returns:In order to draw overall conclusions for the set of events being analyzed, the abnormal return observations must be first aggregated. The aggregation is performed through time and across stocks. By aggregating across all the stocks, we obtain:CAR variance, we assume that N is the total number of events. Finally, we introduce the test statistic τ is the time index inside the event window, and |τ2 − τ1| is the total length of the event window.For the calculation of the shown in ):var(CA


Lung ultrasound is a novel technique for detecting generalized fluid overload in children and adults with end-stage renal disease (ESRD). Echocardiography and bioimpedance spectroscopy are established methods, albeit variably adopted in clinical practice. We compared the practicality and accuracy of lung ultrasound with current objective techniques for detecting fluid overload in children with ESRD.A prospective observational study was performed to compare lung ultrasound B-lines, echocardiographic measurement of inferior vena cava parameters and bioimpedance spectroscopy in the assessment of fluid overload in children with ESRD on dialysis. The utility of each technique in predicting fluid overload, based on short-term weight gain, was assessed. Multiple linear regression models to predict fluid overload by weight were explored.r = 0.57, p = 0.005). A non-significant positive linear correlation was observed between fluid overload by weight and bioimpedance spectroscopy , systolic blood pressure  and physical examination measurements , while a non-significant negative linear relationship was found between the inferior vena cava collapsibility index and fluid overload by weight . In multiple linear regression models, a combination of three fluid parameters, namely lung ultrasound B-lines, clinical examination and systolic blood pressure, best predicted fluid overload .A total of 22 fluid assessments were performed in 13 children  with a median age of 4.0 (range 0.8–14.0) years. A significant linear correlation was observed between the number of B-lines detected by lung ultrasound and fluid overload by weight (Lung ultrasound may be superior to echocardiographic methods and bioimpedance spectroscopy in detecting volume overload in children with ESRD. Given the practicality and sensitivity of this new technique, it can be adopted alongside clinical examination and blood pressure in the routine assessment of fluid status in children with ESRD. Chronic fluid overload contributes to cardiovascular morbidity in children with end-stage renal disease (ESRD) –3. MinimA number of techniques are available to assess fluid overload in adults and children with ESRD, including bioimpedance spectroscopy (BIS) –7, echocIn this prospective observational study, all infants and children (age range 0–18 years) with ESRD receiving dialysis [haemodialysis (HD) or peritoneal dialysis (PD)] in our regional paediatric nephrology center between 1 May 2015 and 1 October 2015 were eligible to participate. Exclusion criteria were co-existent lung fibrosis, atelectasis, lymphangitis, interstitial lung disease, cardiac failure, acute respiratory distress syndrome or congenital cardiac anomalies. In patients with these conditions, pulmonary or cardiac pathology may confound the assessment of generalized fluid overload with lung ultrasound or echocardiography.Thirteen children with a median age of 4.0 (range 0.8–14.0) years ultimately participated in the study, of whom five children received chronic HD and eight received PD for ESRD . The operator was blinded to patients’ BIS and lung ultrasound measurements. Studies were performed in guided B- and M-mode with concurrent electrocardiography, with transducer frequencies appropriate for body size as per the recommendations of the American and European Society of Echocardiography for paediatric patients , 15. ExaBIS was performed in children aged >2 years using a portable whole body BIS device . This device uses 50 alternating current frequencies between 5 and 1000 kHz to measure body composition in relation to total body water, intracellular volume and extracellular volume as reflected by resistance to electric current. BIS was not performed on infants under 2 years of age as the technique has not been validated with appropriate reference algorithms in this age group. One physician took measurements prior to dialysis sessions. Patients were recumbent for at least 5 min prior to measurements, with arms and legs spread at an angle of approximately 30°. Children on PD lay supine with the abdomen drained. Four non-recyclable electrodes were attached to the hand and foot on the non-fistula side. If more than one assessment was undertaken, care was taken to fix electrodes in the same position for each measurement. Measurements with less than 85 % quality were discarded, and the mean of two sequential readings was used for analysis. Excess extracellular water was recorded both in litres and as a percentage of predicted values. Similar to echocardiography, post-dialysis BIS readings were not taken due to the need to wait 2–3 h for vascular refilling ; waitingOur ‘gold standard’ comparator was fluid overload by weight, expressed as the proportional (%) increase from the target weight. The target weight was that prescribed by the responsible physician using clinical data synthesized with trends in nutrition and growth. Systolic blood pressure elevation was expressed as the proportional (%) increase from the 50th blood pressure centile appropriate for age and height . EchocarThe primary outcome was the correlation of technique-specific measurements with the increase in patient weight from baseline. Pairwise correlation between individual fluid overload parameters was determined using Pearson’s correlation. Agreement between fluid measurement techniques and the gold standard comparator, namely weight overload, was assessed using Bland–Altman analysis following natural logarithmic transformation of variables. Multiple linear regression models to predict fluid overload by weight were explored. Optimal variable selection for the regression model was undertaken using the leaps and bounds algorithm . The freA total of 22 fluid assessments were performed in 13 children. Two separate fluid assessments were performed in nine of these children, with each assessment representing different degrees of fluid overload. Single fluid assessments were undertaken in the remaining four patients, all of whom had stable fluid status and residual urine output of >25 ml/kg/day. At each assessment, all fluid-related variables were measured, with the exception of BIS, which was not performed in the two children aged <2 years. One child was obese with a body mass index of >95th centile for age. There were no associated technical difficulties in obtaining adequate lung ultrasound images to assess B-lines.The children were assessed as clinically euvolaemic in 82 % of the physical examinations, as mildly clinically dehydrated in 14 % and as mildly overloaded in 4 %. No children were assessed as having moderate or severe clinical fluid overload at the time of the physical examination. Median fluid overload by weight for all assessments was 0.5 % (range −5.0 to 6.0 %). The median systolic blood pressure elevation above the 50th blood pressure centile was 6.4 % (range −20 to 52 %).2) during inspiration and 1.39 (range 0.6–2.1) cm/(kg/m2) during expiration. The median IVC collapsibility index was 50 % (range 25–83 %). BIS measurements showed a median overhydration of −0.6 % (range −4 to 2.0 %).The median number of B-lines on the lung ultrasound image was four (range 0–26). The presence of up to five B-lines is considered to be a normal variant , 22. Medr = 0.57, p = 0.005; Fig. r = 0.61, p = 0.03) and HD , systolic blood pressure  and clinical examination score . Bland–Altman analysis showed acceptable levels of agreement between B-lines and the gold standard comparator of fluid overload . The final model with beta coefficients is summarized below:A combination of three fluid parameters, namely B-lines, clinical examination and systolic blood pressure, best predicted fluid overload by weight in a multiple linear regression model (The frequency of dissociation between BIS measurements and blood pressure was 36 %, with three of 11 measurements showing underhydration with elevated blood pressure, and one of 11 measurements showing overhydration with low blood pressure Fig. a. For thIn this cohort of 13 infants and children with ESRD, fluid overload was best predicted using a combination of physical examination, blood pressure and lung ultrasound measurements. No significant predictive value from BIS or echocardiographic measurements was observed in the assessment of fluid overload in this study.The BIS data from the present study contrast with previous reports of the utility of this technique in facilitating an accurate assessment of overhydration in children on dialysis , 23, 24.Echocardiography is a well-established technique for fluid overload assessment in children. IVC diameter and collapsibility index were reported to be useful measures of overhydration in 14 children receiving HD as far back as two decades ago . These fThe application of lung ultrasound imaging for the detection of generalized fluid overload in the dialysis population is receiving growing attention. In adult dialysis patients, the technique is reliable in quantifying subclinical fluid overload , 28, 29,Physical examination is the mainstay of fluid assessment in the clinical setting; however, physical examination findings are subject to significant variability, even in the hands of experienced physicians . Our datHypertension is a well-established complication of ESRD in both adults and children . While eThe clinical significance of subclinical volume overload detected using techniques such as BIS remains open to debate in view of significant dissociation between hypertension and overhydration measurements in children . Our datThis study has a number of limitations. As a relatively small, single-centre study, it lacks the statistical power required to reach firm conclusions on the superiority of any fluid assessment technique. The study population is heterogeneous with respect to both patient age and dialysis modality. The gold standard measure of fluid overload, namely deuterium dioxide dilution, was not used due to ethical concerns about repeated blood sampling in infants and children. Notwithstanding these limitations, sufficient data relating to the practicality and utility of fluid assessment techniques are presented which allow their use in paediatric patients with ESRD to be compared for the first time.The results of this study suggest that lung ultrasound imaging may be superior to both echocardiographic methods and BIS in detecting volume overload in children with ESRD. Our data demonstrate that lung ultrasound was the most practical of the three techniques, being undertaken with a portable device at the bedside in <5 minutes per examination. The technique is straightforward to learn for non-radiologists. It can be used in infants and babies under 2 years of age, for whom BIS is not validated. Given the practicality and sensitivity of lung ultrasound imaging, this technique can be adopted alongside clinical examination and blood pressure in the routine assessment of fluid overload in children with ESRD.


Drosophila morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the nullo gene product. We further show that checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis.Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early  One of the primary events that must occur repeatedly throughout a complex animal’s lifetime is the ingression of a plasma membrane furrow. Furrow formation and ingression are requisite elements of cell division, and drive the physical separation of one cell into two cells. However, the mechanisms that permit an embryo to change the length and size of a furrow are unclear. Here, we show that a combination of higher ingression rates and longer duration phases drive changes in furrow dimensions through the introduction of a new ingression phase. These changes are driven by the embryo’s own genome, and suggest that zygotic transcription controls organismal form at an earlier time point than previously appreciated. Additionally, the failure to properly lengthen furrows as development proceeds causes defects in chromosome segregation during cell division and results in massive genomic instability. Our data demonstrate the importance of the dynamic regulation of furrow dimensions to organismal form and viability. Furrow ingression is an obligatory step in animal cells during cell division, and is a critical mechanism that underlies the ability of animal cells to divide and provide new cells for tissue homeostasis and development. While much of our knowledge of how plasma membrane furrows form and ingress comes from studies in isolated tissue culture cells, cells in different tissues and developmental contexts complete cell division at different rates, and possess different constraints in the resources available to them [reviewed in Drosophila embryogenesis initiates with a single nucleus that begins 13 cycles of rapid nuclear replication and division in an acellular syncytium. The first nine rounds of nuclear divisions occur deep in the yolk, with nuclei migrating to the periphery of the embryo at cycle 10. At this point, the density of nuclei and their arrangement in a common cortical plane requires four cycles of transient plasma membrane furrow formation  to adequately partition mitotic figures and ensure genomic stability [tability . At cycltability –8. It isDrosophila embryo. However, the function of these early transcripts as well as the clearance of maternal products in directing morphogenesis prior to cycle 14 has not been clear. Here, we examine the extent to which the ingression of differentially dimensioned furrows in the early embryo is a result of MBT-based developmental regulation of furrow dynamics.Intriguingly, it is in this same period of rapid furrow morphogenesis that the zygotic genome becomes transcriptionally active, and a hand-off in genetic regulation occurs from maternal to zygotic control . While the MBT is often classically-defined by events that occur during cycle 14, zygotic gene expression can be detected as early as cycle 2 at a few isolated loci , and sevDrosophila embryo, we used live 4D imaging to follow furrow formation and retraction behaviors. We find that a new ingression phase (Ingression II) follows the stabilization phase of cycles 12 and 13 and helps to drive the four-fold lengthening of furrows that occurs between cycles 10 to 13. While Ingression I is largely dependent on maternal gene products, Ingression II requires zygotic gene transcription. Either genetic or pharmacological blockage of new transcript production results in a complete loss of Ingression II and a loss of furrow lengthening. Through the use of compound chromosomal fly lines, we show that multiple loci on the X, II, and III chromosomes are required for the developmental production of differentially dimensioned furrows. We also find that cell cycle regulation of interphase and mitotic periods, which occurs during cycles 10–14, is permissive for furrow regulation, but is not strictly deterministic. Thus, we propose that direct developmental regulation by zygotic products is responsible for furrow lengthening and the changes in furrow dimensions in the early Drosophila embryo.To perform the first, comprehensive time-resolved measurements of wild-type syncytial furrow dynamics in the early At nuclear cycle 10 the majority of nuclei have migrated to adopt a subapical position beneath the cortex. At this point, five cycles of plasma membrane furrow formation begin, with furrows of characteristic differing lengths forming in each cycle. To examine this more closely, we imaged living embryos expressing markers of the plasma membrane (Gap43:mCherry) and chromosomes (Histone:GFP), which permits the imaging and quantitation of individual furrow dynamics, as well as the tracking of cell cycle behaviors. The measured, true furrow region is defined as the furrow length that stretches from where apical caps meet to the end of the furrow canal . To estaSimilar to previous results , 22, furThe lengthening of furrows raises the question: what are the important formative events that drive changes in furrow dimensions? Between cycles 10 and 11, furrow invagination is driven by a single, initial ingression phase (Ingression I), which is then followed by a period of stable furrow lengths and eventual furrow retraction . Furrow Interestingly, as furrows begin to reach depths 3x (cycle 12) and 5x (cycle 13) greater than initial furrow lengths at cycle 10, a different dynamic is initiated. Furrow lengthening during cycles 12 and 13 becomes dependent on the introduction of a new ingression phase (Ingression II) . IngressNotably, the maximum and average ingression rates of Ingression I from cycle 10–14 are largely similar, although the average rate increases by an additional 0.6x at cycle 13 . HoweverDuring cycles 10 through 12, the duration of the stabilization phase stays at approximately 3.5 minutes despite the lengthening of the furrow cycles. At cycle 13, stabilization increases to 5.1 minutes , howeverPrevious work has shown that an actin-dependent apical budding process begins at cycle 10 when nuclei assume a subcortical position , 23. GivThe activation of zygotic gene expression occurs in the same temporal period as the rapid changes in furrow dimensions. To examine if zygotic guidance of furrow behaviors directs furrow morphologies, we injected embryos with α-amanitin, a drug that selectively inhibits RNA polymerase II , 25. Strzelda. Regarded as a master regulator of zygotic genome activation (ZGA), zelda is a transcription factor required for the expression of a wide portion of early zygotic genes (zelda-dependent genes) [zelda mutant embryos Ingression II is almost entirely lost, although furrows proceed ~1μm deeper than in α-amanitin embryos  , 26, 27. embryos . In addi embryos , and Ingntinuity . The slizelda mutant embryos are either longer or very similar to wild-type cycle times . However, despite this elongation of metaphase, Ingression II is still absent. Similarly, zelda mutant embryos have a shortened interphase, but possess a deeper Ingression I , and then appear incapable of further ingression , smg appears to have the earliest function in maternal clearance [smg mutant alleles to examine the effects of maternal gene decay on furrow dynamics. smg mutant embryos do not show gross disruptions of furrow behaviors of the kind observed in α-amanitin, zelda, compound-X, or mei41 compromised embryos. smg embryos have similar furrow behaviors and display dynamics comparable to wild-type embryos  is an essential factor for maternal mRNA destabilization , 20. Whilearance , 36. We  embryos . Howevercycle 14 ,19,21.2 at cycle 10 to 15.4 nuclei/1000μm2 at cycle 13  and 16.2% (zelda) of nuclei becoming polyploid through fusion events and/or failures in chromosomal segregation  a direct model, in which shorter cell cycle times limit the periods that allow furrow ingression, or 2) an indirect model, in which the failure of checkpoint function disrupts the amount of zygotic transcription that can occur. As furrows in mei41 mutants plateau several minutes before anaphase and the usual termination of Ingression II, we favor the second model in which products essential to furrow ingression are not made at high enough levels to support a continued furrow ingression during metaphase of cycle 13. This would also be consistent with data from α-amanitin treated embryos in which the cell cycle is longer than in wild-type embryos, but furrow lengths are shortened. Additionally, although furrow processes often have a general correspondence to phases of the cell cycle, in various mutant and small molecule-treated backgrounds these associations break down. These results suggest that cell cycle elements that regulate interphase and mitotic periods have a permissive, rather than strictly deterministic, effect on furrow dynamics.Many higher organisms, including M phases –47. As tle 12–14 . Our worFurrows progress from 1.5μm at cycle 10 to 8μm at cycle 13 and 28μm at cycle 14. This deepening of furrows is associated with an increase in nuclei number, and suggests a functional role for furrows in maintaining proper chromosomal segregation. Indeed, throughout the early cell cycles there is an inverse relationship between furrow length and mitotic defects. This mirrors data for furrow-less embryos, in which chromosomal segregation defects are apparent from cycle 11 through cycle 13, although cycle 10 embryos are relatively free of mitotic defects [zelda mutant embryos, ~95% of segregation defects are through adjacent chromosomal fusions. However, the mitotic collapse phenotype could be the result of either a lack of furrows to properly anchor and attach the mitotic spindle, or could be a result of cell cycle defects that cause tangled chromosomal complements that inhibit separation. Previous work on furrow-less embryos generated by defects in membrane trafficking has shown that both the adjacent fusion and mitotic collapse phenotypes occur in embryos that still possess otherwise wild-type cell cycle times and behaviors [Live imaging of chromosomal dynamics also permitted the tracking of individual mitoses, and demonstrated that defects in segregation occur through two possible events: 1) the fusion of adjacent chromosomal complements, usually after the successful completion of anaphase, or 2) the collapse of individual mitotic figures  Fig 7C;. The fusehaviors . It alsoehaviors , 50. As o C. The following stocks were used in this study: Gap43:mCherry , P{OvoD1-18}, P{neoFrt19A}/C(1)DX/Y; P{hs-Flp} , and Sqh-GFP:moeABD . Resille:GFP (plasma membrane and furrow marker), Spider:GFP (plasma membrane and furrow marker), His2Av:GFP and His2Av:RFP , Df(1)Sxl-bt (nullo Df)/FM7 , smg1/TM3 , and Df(3L)ScfR6/TM3  were provided by the Bloomington Drosophila Stock Center. zld294 P{neoFRT19A} , mei41D3, mei4129D , C(1)DX, C(2)v, and C(3)Se were kindly provided by the Wieschaus lab (Princeton University).All fly stocks were maintained at 25zelda germline clones were generated by crossing zld294 FRT19A females to ovoD, FRT19A males. Larvae were heat shocked three times for 2 hours over the course of 5 days to induce recombination events.nullo, halo, or twist/eve/Kr, respectively. For compound III analysis, furrow dynamics were followed by extracellular dextran488 injection. Aneuploid 3L and 3R embryos were determined by fuzzy-cellularization, and Serendipity-α, respectively.To image furrow dynamics in aneuploid backgrounds, Compound X females were crossed to males carrying both membrane and histone markers. To create a Compound II stock carrying both membrane and histone markers, we took advantage of the fact that autosomal, double balancer stocks  have a low rate of missegregation defects that generate aneuploid gametes. Sp/CyO; His2Av:RFP, Spider:GFP/TM3 females were crossed to Compound II C(2)v males. Rare F1 males (C(2)v; His2Av:RFP, Spider:GFP/+) were backcrossed to compound stocks and embryos from the F2 progeny were used for imaging. Aneuploid X, 2L or 2R embryos were determined by scoring for phenotypes caused by deficiency of A spinning-disk confocal microscope from Zeiss/Solamere Technologies Group with 63X/1.4NA objective lens was used for time-lapse imaging. The embryos were collected on standard yeasted apple juice agarose plates, dechorionated, and transferred to an air-permeable membrane in Halocarbon 27 oil (Sigma). A coverslip was placed on embryos for live imaging. For individual time-lapse imaging, full z-stacks were acquired at either 20s or 30s intervals. Each z-stack was comprised of 30–33 z-slices at a 0.5μm interval. For cytoplasmic budding imaging, 30 z-slices with a 0.3 μm interval were taken at every 20s. All movies were acquired at 25°C.Embryos were dechorionated in 50% bleach solution and fixed for 20 minutes at the interphase of heptane and either 18.5% formaldehyde (Electron Microscopy Sciences) (for anti-Nullo staining), or 4% formaldehyde (for anti-Lamin staining) in 0.1 M sodium phosphate buffer (pH 7.4). Then the embryos were manually devitellinized and stained with Alexa 546-phalloidin , mouse anti-Lamin , or mouse anti-Nullo . Secondary antibodies conjugated with Alexa 488 (Molecular Probes) were used at 1:500. Embryos were mounted in ProLong Gold with DAPI staining (Life Technologies). Immunostained embryos were imaged with an Olympus Fluoview FV100 confocal laser scanning microscope with 40X or 60X 1.35NA objective lens. Images were acquired using 12 ms/pixel exposure settings.Embryos were glued on a coverslip after dechorionation. The embryos were dehydrated for 12–15 min, covered in Halocarbon oil 700 (Sigma), and injected with α-amanitin , Dextran-Alexa488 , or water. After injection, the embryos were placed on an air-permeable membrane and imaged on the spinning disk confocal microscope.http://www.flyrnai.org/snapdragon) to decrease potential off-target effects. dsRNA was made using Mega script T7 Transcription Kit (Ambion) and purified by Quick-RNA Microprep kit (Zymo Research). The concentration of dsRNA was determined by a NanoDrop ND1000 spectrophotometer (2000 ng/μL). For injection of siRNA, the embryos were prepared in the same method as drug injection. After injection, embryos on a coverslip were immersed in Halocarbon 27 oil (Sigma) and placed on a gas-permeable membrane and imaged on the spinning disk confocal microscope.Primers for siRNA treatments were designed using the SNAPDRAGON RNAi design program  Wild-type furrow dynamics, including cellularization, during cycles 10–14 . (B) Furrow morphology during cycles 10–13. Scale bar = 5 μm. (C) Wild type furrow dynamics from cycle 12. Red line shows the furrow dynamics without stabilization phase (n = 5 embryos); and black line shows with stabilization phase (n = 7 embryos). (D) Cycle 13 furrow dynamics for different membrane markers and injection controls (n≥3). (E) Apical actin cap dynamics (GFP:moeABD) and cell cycle (His:RFP) during the metaphase of cycle 10 to the interphase of cycle 11. Z-layer of actin and nucleus is 1 μm and 5 μm below the vitelline membrane, respectively. (F) Apical actin cap initiation (GFP:moeABD) and furrow dynamics (Gap43:mCh) at the end of cycle 11. (G) Apical actin displacement during cycle 10–13 (n = 4).(TIF)Click here for additional data file.S2 Fignullo Df furrow morphology in cycle 13, and z planes at -1 μm, -3 μm, -4 μm, -5 μm, -6 μm, -7 μm, and -8 μm. Scale bar = 5 μm. (B) Wild type, intact and deepest Aneuploid X and nullo Df furrow length for cycle 12 and 13 . (C) Nullo protein localization in wild-type and nullo Df embryos in cycles 12–14. Anti-Nullo (green channel), F-actin , and merged channel. Scale bar = 5 μm. (D) Wild type, intact and deepest Aneuploid X and nullo Df maximal furrow ingression rate in cycle 12 and 13 .(A) Aneuploid X and (TIF)Click here for additional data file.S3 Figns: not significant. (B) Average furrow ingression I rates of WT, aneuploid 3L, and 3R embryos during cycles 11–13 (n≥3). *:p<0.05; ns: not significant. (C) Chromosomal morphologies indicate cell cycle status (Histone:GFP) at 0 min (interphase), 4 min (prophase), 8 min (metaphase), 10 min (anaphase), 12 mi (telophase), and 13 min (start of new cell cycle). Example nucleus from cycle 12, but similar morphologies are present during cycles 10–13. Arrowhead indicates the bright puncta in nuclei during prophase. Scale bar = 5 μm. (D) Furrow traces from individual mei-41 mutant embryos demonstrate variable Stabilization timing and period, but possess reduced, separable Ingression I and Ingression II phases. (E) Furrow dynamics for zld RNAi (black curve) and zld RNAi + mei41 mutation (blue curve) in Spider:GFP, His:RFP background (n = 4). (F) Maximal furrow length in zld RNAi and zld RNAi + mei41 mutation during cycle 11–13 (n = 4). *: p<0.05; ns: not significant. (G) Maximal furrow ingression rate of zld RNAi and zld RNAi + mei41 mutation during cycle 11–13 (n = 4). *: p<0.05; ns: not significant. (H) Nuclear envelope staining in WT and zld mutant in cycle 13. Anti-Lamin (green channel), DAPI (blue channel), and merged channel are shown. Scale bar = 5 μm. (I) Adjacent nuclear fusion (upper panels) and mitotic nuclear fusion (bottom panels) phenotypes in α-amanitin injected embryo during cycle 13. Prophase, metaphase, anaphase, telophase and interphase in the next cycle are shown. Arrowheads indicate missegregated chromatins. Scale bar = 5 μm. (J) Intact furrow length and mitotic defects. The percentage of mitotic defects at cycle 13 in α-amanitin injected, zld, Aneuploid X, nullo, and WT embryos is plotted, as well as the intact furrow lengths during metaphase in cycle 13. (K) Interphase nuclear densities during cycles 10–13 (n = 4).(A) Average furrow ingression I rates of WT, aneuploid 2L, and 2R embryos during cycles 11–13 (n≥3). *:p<0.05; (TIF)Click here for additional data file.


Innate lymphoid cells (ILC) are part of a heterogeneous family of haematopoietic effector cells which lack re-arranged antigen-specific receptors. They promote host defense and contribute to tissue and metabolic homeostasis, wound healing and immune surveillance. Their role in human cancer immunity is less defined, and therefore we aimed to identify the frequency and phenotype of distinct ILC groups in various types of cancer.Tissue samples and peripheral blood were collected from patients undergoing surgical resection of gastrointestinal and breast tumours. Single cell suspension of tumour tissue was immediately obtained following surgery using tumour dissociation.p value: 0.04) in malignant breast cancer tissue and significantly higher frequencies of group 1 ILC  in malignant gastrointestinal tumours. Tumour infiltrating ILC were found to show an activated phenotype with higher expression of MHC-II, KLRG1, early activation marker CD69 and CD44.We observed significantly higher frequencies of ILC2 (Activated innate lymphoid cells infiltrate tumours dependent on tumour type and location.The online version of this article (10.1186/s12885-018-4262-4) contains supplementary material, which is available to authorized users. Significant advances have been made in understanding the immune surveillance mechanisms in identifying and destroying tumour cells , 2. The + and NCR− ILC3s. LTis are essential for generation of lymph nodes, Peyer’s patches and isolated lymphoid follicles. Similar to Th17 and Th22, ILC3s produce IL-17A and IL-22 [Another cell population that has been implicated in immune defence against tumour cells are natural killer (NK) lymphocytes. Infiltration of NK cells is a recognised feature of most cancers , 7. Unlind IL-22 , 13.− NK1.1+ CD49ahi cells in mammary tumour tissue of MMTV-PyMT (PyMT) mice [−/− mice when infected with H.hepaticus and treated with the carcinogen azoxymethane (AOM) [− IL-22+ cells have been observed in the tumour tissue of patients with CRC [Given that ILC are mainly located in pathogen entry sites e.g. mucosal surfaces of gastrointestinal tract, respiratory system and the skin, their main function was considered to be protection against infections. However, recent data show that ILC also contribute to immunity against cancer in mice . Dadi anMT) mice . Due to MT) mice . In anotne (AOM) . Furtherwith CRC .− Sca-1+ ST2+ innate lymphoid cells (ILC) and reduction of NK cells in 4 T1 breast cancer model following administration of IL-33. IL-33 promoted tumor growth, development of lung and liver metastases, intratumoral cell proliferation and neovascularisation [ILC2 and ILC2 related genes  were increased in the peripheral blood of patients with gastric cancer compared to healthy controls [risation .While ILC family sub-populations have been investigated in animal models, the role of these cells in protection against human malignancy has been less explored. Nonetheless the potential contribution of these cells has been reviewed extensively –23. HereSamples were obtained from 13 patients with gastrointestinal tumours, 16 patients with malignant breast cancer and 5 patients with benign breast tumours that underwent surgical removal of tumour tissue . Solution A, Solution B and Solution C from tumor dissociation kit were dissolved by sterile PBS solution and aliquoted into small tubes stored at − 20 degrees. Briefly, samples were cut into small pieces, (1-3 mm) and transferred into C tubes (Miltenyi Biotec 130–096-334). Before loading the C tube into gentleMACS Octo-dissociator (Miltenyi Biotec 130–096-427), 1 unit of Solution A, Solution B and Solution C were added into C tube containing 10 ml RPMI1640. The tubes were then run on ‘h_tumor_01’ and ‘h_tumor_02’ programmes respectively with intervals of 30- min incubation at 37 ͦC with rotation. Following second incubation the tubes were run on ‘h_tumor_3’ programme and samples passed through 70 μm strainer. The cells were centrifuged and washed with RPMI 1640 medium. The enzymatic degradation steps were removed for dissociation of gastrointestinal tumours as this did not render any benefits in terms of cell yield. PBMCs were isolated from whole blood samples using ficoll density gradient centrifugation.The following anti human antibodies were purchased from Biolegend unless stated otherwise. CD19 , CD11b , CD11c (BU15), FcεRI (AER-37 (CRA-1)), CD14 , TCRαβ (IP26), TCRγδ (B1), CD3 (SK7 and OKT3), CD45 (H130), CRTH2 , CD127 (A019D5), c-Kit (104D2), CD44 (G45–26 and IM7), CCR7 (G043H7), PD-1 (EH12.2H7), CTLA-4 (L3D10 and BNI3 BD Biosciences), CD56 (HCD56 and 5.1H1L), CD69 (FN50), KLRG1 (SA231A2), MHC II (L243) and LIVE/DEAD Fixable Violet Dead Cell Stain Kit. Lineage stains were combined through a single channel, which allowed ILC phenotypic marker assessment but limited the potential to correlate with other cell populations.Samples were acquired using FACSDiva software on LSR Fortessa (Becton Dickson). The flow cytometry data were analysed using FlowJo software.− c-Kit−), group 2 ILC (CRTH2+ c-Kit +/−) and group 3 ILC (CRTH2− c-Kit+)  of ILC2 were observed in malignant cancer tissue compared to benign tissue  in malignant breast cancer tissue , CD45, CRTH2, IL-7Rα and c-Kit. Lineage negative cells expressing haematopoietic and lymphoid markers CD45 and IL-7Rα were selected and based on the expression of CRTH2 and c-Kit were sorted into group 1 ILC  and NK cells  were accumulated in the malignant tissues, whereas smaller numbers of ILC3s  were found in the tumour tissues  and Cytotoxic T-Lymphocyte Associated protein (CTLA-4) are inhibitory receptors that modulate immune responses and attenuate tissue damage . Tumour 9 and Cytp value: 0.04) and NK cells  compared to circulating ILC in peripheral blood  and ILC3  populations in GI tumours compared to para lesional tissue, while CTLA-4 was not significantly altered -II, CD69, CD44, Killer cell lectin-like receptor G1 (KLRG1) and CCR7 in peripheral blood, benign and malignant breast tumour tissue and malignant and para-malignant tissue in GI cancers  and malignant  breast tumour tissue express significantly higher level of HLA-DR compared to peripheral blood . Around 20%–40% of T cells in young adults can express this marker  which rihi group 2 innate lymphoid cells, designated inflammatory ILC2s ‘iILC2’ was discovered in mice following infection. These cells showed more plasticity than KLRG1int cells and could give rise to IL-17A producing ILC3s [ILC populations, in mice and human, express KLRG1 receptor which is an indicator of activation. A population of IL-25 responsive KLRG1ng ILC3s . Higher ng ILC3s . KLRG1 eng ILC3s  and colong ILC3s .p values ILC1: 0.0001, ILC2: 0.005, ILC3: 0.0002, NK: 0.0001) and breast tissue  of patients with malignant breast tumour express KLRG1 compared to patients with benign tumours, consistent with a more activated phenotype  and ILC2  populations in malignant GI tumour tissue express significantly elevated KLRG1 compared to paramalignant tissue  [IFN-γ is the cardinal cytokine produced by ILC1 and it is a critical regulator of anti-tumour responses. The anti-proliferative activity of IFN-γ is mediated by activation of STAT-1 and induction of p21 4 (CDK) . IFN-γ/S 4 (CDK) . Product 4 (CDK) . The ant 4 (CDK) . Here weOn the contrary to ILC1 and NK cells, cytokines that activate group 2 ILC may be more protumourigenic. For example, IL-33 promotes chronic inflammation and tumour development in some models. Serum levels of IL-33 are increased in breast, lung, gastric and hepatocellular carcinomas. Blocking IL-33 receptor (ST2) reduced generation and size of tumours in a colon cancer model . In addi−/− mice infected with H.hepaticus and treated with the carcinogen azoxymethane (AOM) develop IL-22 dependent colorectal cancer. Colonic ILC3s are thought to be the source of IL-17A and IL-22 and are found in higher frequencies in this model [Both pro- and anti- tumour properties of group 3 innate lymphoid cells have been observed. 129SvEv.RAGis model . In contis model .Comparing tumour infiltrating ILC populations with circulating ILC, showed that ILC populations have an activated phenotype in tumour tissue with higher expression of MHC-II, KLRG1, CD69 and CD44. Unlike adaptive immune responses, ILC populations lack RAG-dependent rearranged antigen receptors. They can be activated by direct interactions mediated by pattern recognition receptors (PRR) and natural cytotoxicity receptors (NCR) or indirectly by activating cytokines. Tumour associated ILC are an early source of innate cytokines. Direct interaction with cancer cells and inflammatory tumour microenviroments may contribute to their activated phenotype. Here we present an observational study, but it will clearly be important to proceed to define the underlying mechanistic associations.Functional diversity of ILC populations combined with expression of similar activatory/ inhibitory receptors make it difficult to extrapolate the use of blocking or activatory agents. For instance blocking inhibitory receptors KLRG1, PD1 and CTLA-4 on ILC1 population may help in increasing production of anti-tumour cytokines, IFN-γ and TNF-α, but as these receptors are also expressed highly on ILC2 population in tumour environments, using blocking agents might contribute to increase in type 2 cytokine production and modulate tumour growth. Further studies are crucial to find strategies to specifically target ILC subpopulations to define underlying mechanisms and new approaches to treatment.Most studies on the role of innate lymphoid cells in cancer have been done on animal models. Therefore, further clinical investigations in humans are required to define the extent of their role in cancer. It is likely that not only the intrinsic ability of ILC but also the nature of transformed cells and tumour microenvironment are essential determinant factors. Further understanding of their role provides novel targets in immunotherapeutic strategies to treat cancer.Innate lymphoid cells infiltrate tumours and show altered activation status. These findings would be consistent with a role for innate lymphoid cells in disease or its control. Further understanding of mechanism may contribute to future therapeutic advance.Additional file 1:Figure S1. Representative staining of ILC populations. ILC were gated on lineage negative, CD45+, CD127+ cells and based on the expression of CRTH2 and c-Kit divided into ILC1 , ILC2  and ILC3 . Figure S2 Representative example of ILC1, ILC2, ILC3 phenotypic analysis from benign breast tissue. Figure S3 Representative example of ILC1, ILC2, ILC3 phenotypic analysis from malignant breast tissue. Figure S4 Representative example of ILC1, ILC2, ILC3 phenotypic analysis from malignant GI tumour tissue. Figure S5 Representative example of ILC1, ILC2, ILC3 phenotypic analysis from paralesional GI tumour tissue. Table S1 Patient characteristics. (PDF 5287 kb)


The Ki-67 Labelling Index (LI) is used as an ancillary tool in glioma diagnostics. Interobserver variability has been reported and no precise guidelines are available. Nor is it known whether novel digital approaches would be an advantage. Our aim was to evaluate the inter- and intraobserver variability of the Ki-67 LI between two pathologists and between pathologists and digital quantification both in whole tumour slides and in hot spots using narrow but diagnostically relevant intervals.In samples of 235 low and high grade gliomas, two pathologists (A and B) estimated the Ki-67 LI  for whole tumour slides and for hot spots. In 20 of the cases intraobserver variability was evaluated. For digital quantification (C) slides were scanned with subsequent systematic random sampling of viable tumour areas. A software classifier trained to identify positive and negative nuclei calculated the Ki-67 LI. The interobserver agreements were evaluated using kappa (κ) statistics.The observed proportions of agreement and κ values for Ki-67 LI for whole tumour slides were: A/B: 46% (κ = 0.32); A/C: 37% (κ = 0.26); B/C: 37% (κ = 0.26). For hot spots equivalent values were: A/B: 14% (κ = 0.04); A/C: 18% (κ = 0.09); B/C: 31% (κ = 0.21).Interobserver variability was pronounced between pathologists and for pathologists versus digital quantification when attempting to estimate a precise value of the Ki-67 LI. Ki-67 LI should therefore be used with caution and should not be over interpreted in the grading of gliomas. Digital quantification of Ki-67 LI in gliomas was feasible, but intra- and interlaboratory robustness need to be determined.The online version of this article (10.1186/s13000-018-0711-2) contains supplementary material, which is available to authorized users. Gliomas are the most frequent primary brain tumours, of which glioblastomas are the most malignant . AccordiThe proliferation marker Ki-67 or MIB-1) has been suggested as an ancillary marker  has been in the gSeveral methods, including digital approaches, have been proposed to evaluate the Ki-67 LI in gliomas and other neoplasms of the central nervous system –14. In gThe aim of this study was to evaluate the inter- and intraobserver variability of the Ki-67 LI between two pathologists and between pathologists and a digital quantitative approach. The pathologists used a semi-quantitative approach. Both whole tumour slide mean values and hot spot values were evaluated for both approaches.A poster with preliminary results has been presented .The study was based on a cohort of adult patients with primary glioma with no previous treatment diagnosed between 2005 and 2009. The cohort has been used in various biomarker studies –22. FormFor immunohistochemical staining a 3 μm section was cut from each tissue block. All stained sections were whole tumour sections. The immunostaining was performed on the automated immunohistochemical staining system BenchMark Ultra , and all sections were stained in the same run or in runs following one another. All reagents used were from Ventana Medical System, Inc., USA. Antigen retrieval was achieved using heat induced epitope retrieval for 48 min with a maximum temperature of 100 °C and Cell Conditioner 1 as the buffer. The primary antibody used was monoclonal rabbit anti-Ki-67 (30–9), ready to use dilution, with an incubation time of 12 min at 36 °C. The visualization system was OptiView DAB and counterstaining with Hematoxylin II and Blue Reagent followed immunostaining. Nuclear staining was considered positive. Appropriate staining of the germinal centres and squamous epithelium of normal tonsil served as a positive control, with superficial epithelial cells representing a negative tissue control. Moreover, omission of the anti-Ki-67 antibody served as a negative control.Two pathologists made a visual estimate of the Ki-67 LI using a light microscope, resembling daily practice. No fixed number of nuclei was counted. This approach was used in order to reflect daily diagnostics. The Ki-67 LI was defined as the percentage of positive tumour nuclei of all tumour nuclei. Necrosis, normal brain tissue, zones of infiltration, endothelial and inflammatory cells were omitted from the evaluation. A whole slide mean value and a hot spot value were estimated for each tumour in independent sessions.To estimate the whole slide mean values, the tumour slides were viewed using the scanning objective (40× magnification) with subsequently evaluation at higher magnification of selected foci of viable tumour areas (100× magnification). The number of selected foci was not defined beforehand in order to reflect daily diagnostics. Each pathologist chose how many microscopic fields to examine in each case. This number varied between cases depending on the area of the tumour tissue on the slide and the heterogeneity of the tumour tissue.2) at 400× magnification. Selection of hot spots was done by scanning the slide at low magnification, and the hot spot was selected in the area with the highest LI. In each case one hot spot was chosen for evaluation.A hot spot was defined as the area with the most intense proliferation covering an area of at least one high power field . The Visiopharm integrator system software  was used for the digital image analysis. Tumour tissue was outlined manually and necrosis, vessels, invasion zones and normal brain tissue were excluded. Images to be quantified were obtained by systematic random sampling of 5% of the tissue  Ki-67 LI for the two pathologists (A and B) and for the digital approach (C) were: A: 10% [0–60%]; B: 15% [0–50%]; C: 14% [0–51%] Tables . The paiThe median [range] Ki-67 LI for pathologists (A and B) and for the digital approach (C) were: A: 20% [0–90%]; B: 30% [0–80%]; C: 31% [0–81%] Tables . The paiFor the two pathologists there was agreement for A in 13/20 cases  for pathologists. Because of the small number of cases no statistical analysis was done. The sampling of the tissue on the slide was performed by systematic random sampling and thus different sample areas could be quantified when repeating the evaluation. This can explain why the difference of the Ki-67 LI exceeded 5% in two cases. A preliminary test considering reproducibility and time consumption resulted in the choice of a sample fraction of 5% in the present study. In the preliminary test there was no remarkable improvement in terms of reproducibility when using a larger sample fraction except in cases with only little amount of tissue on the slide. Using a larger sample fraction of the digitalised slide or quantifying all the viable tumour areas would be very time consuming because of the large number of sample images per slide combined with the manual step of outlining tumour tissue. In previous studies various digital techniques have been found to be feasible in the determination of the Ki-67 LI in gliomas and meningiomas , 13, 29,In conclusion our results suggest marked interobserver variability when attempting to estimate precise values for the Ki-67 LI. We find that such precise values should be used with caution and such values should therefore not be over interpreted in the grading of gliomas. The digital quantification was feasible, but intra- and interlaboratory robustness of the method need to be determined.Additional file 1:Table S1 Intraobserver variability for pathologist A - Whole tumour slide mean values of Ki-67 LI. Table S2 Intraobserver variability for pathologist B - Whole tumour slide mean values of Ki-67 LI. Table S3 Intraobserver variability for pathologist A – Hot spot values of Ki-67 LI. Table S4 Intraobserver variability for pathologist B – Hot spot values of Ki-67 LI. Table S5 Intraobserver variability for the digital quantification of the Ki-67 LI (%). (DOCX 27 kb)The results of the intraobserver variability.


Auricular hematoma is a frequent complication of blunt trauma to the auricle. These are usually caused by sports such as boxing and wrestling. Hematoma formation is within the space between perichondrium and cartilage on the anterior ear. Because the blood supply to cartilage is dependent on the perichondrium, infection and cartilaginous necrosis often complicate untreated auricular hematomas. If auricular hematoma is not treated properly, it can progress to abscess formation, chronic scarring and fibrous organization with ear deformity “cauliflower” due to a stimulation of mesenchymal cells in the perichondrion to produce a new cartilage. After incision and evacuation of the fluid, a pressure dressings is traditionally applied to prevent reaccumulation in the resultant dead space. Optimal immobilization of the skin over underlying cartilage for 7-10 days prevent the reaccumulation of fluid with subsequent thickening and fibrosis. We report a case of a 40-year-old football coach presented to the Emergency Department with a painless right ear swelling occurred after auricular trauma during training. The clinical examination showed a right fluctuant non-inflammatory swelling of the lateral face of the pinna. Otoscopy and retroauricular region were normal. The needle puncture brought a serohematic fluid and the diagnosis auricular hematoma was made. The collection was drained via a double anterior incision under local anesthesia and a tie through dressing was used. After 10 days, the sutures and tie through dressing were removed. One year after the operation, the ear appeared almost normal.


Affiliation The Acknowledgements section incorrectly spells the institution: Universiti Malaysia Pahang. The Acknowledgements section should read as follows: This research was supported by the University Malaya with Grant No. RP018B-13AET. The authors would like to thank the Centre of Manufacturing System Integration (MSI) and Centre for Product Design and Manufacturing (CPDM) of University Malaya for their support. The authors wish to express their gratitude to the Universiti Malaysia Pahang for funding for the publication (Grant No. RDU140107).