Recovery of active force with low-frequency stimulation is slow and persistent fatigue results from low [Ca2+]m. However, the consequences of intermittent submaximal contractions are not known. The aim of the present study was to investigate the changes in [Ca2+]m and active force during intermittent submaximal contractions and subsequent recovery. Single fibers of mouse flexor digitorum brevis muscles at 32 °C were stimulated with 40 or 50 Hz, for 350 ms every 2 s for 2 min and then every 1 s until < 40% of initial force. Values obtained during the intermittent stimulation were compared with a control force-[Ca2+]m relationship. A “P”-shaped pattern in the force-[Ca2+]m relationship was observed during intermittent stimulation. Early in the intermittent stimulation, [Ca2+]m increased while active force decreased. Subsequent force potentiation was accompanied by increased Ca2+ sensitivity. Later, as active force declined, [Ca2+]m decreased significantly (p < 0.001). This was followed, in the final phase, by a significant decrease in Ca2+ sensitivity determined by [Ca2+]m at half-maximal force (Ca50) (p = 0.001). Low-frequency fatigue persisted during recovery while Ca50 was not significantly different from prefatigue (p > 0.5). In conclusion, the main mechanism of fatigue is due to decreases in both [Ca2+]m and Ca2+ sensitivity following the initial force potentiation. The intermittent submaximal contractions resulted in persistent low-frequency fatigue seen during recovery, which was explained by depressed [Ca2+]m with no change in Ca2+ sensitivity.Fatigue of single mouse fibers during repeated high-frequency stimulation results initially from decreased Ca However, the pattern of change in [Ca2+]m is not known for intermittent low-frequency stimulation in mammalian muscles, a situation that is more compatible with in vivo muscle activation.The [Cavailable . We alsotivation  and that fatigue . On the 2+]m and some will affect force by altering Ca2+ sensitivity, but either way, changes to both [Ca2+]m and Ca2+ sensitivity will particularly affect low- as opposed to high-frequency force since contraction resulting from this stimulation lie on the steep portion of the force-[Ca2+]m relationship.Activation of muscle for locomotion and general movements typically does not require maximal activation. A common fatigue protocol that takes this into consideration was first used by Burke et al.  for the 2+ sensitivity [2+]m. The mechanism of potentiation is generally thought to be dependent on increased Ca2+ sensitivity in association with regulatory light chain (RLC) phosphorylation. This RLC phosphorylation is thought to increase the rate of attachment of cross-bridges due to the increased mobility of the myosin heads [2+]m. This form of potentiation is effective up to a frequency of stimulation corresponding to that which yields 60% of maximal active force m. Common mechanisms of change in cross-bridge kinetics affecting Ca2+ sensitivity include temperature, pH, myosin RLC phosphorylation, [Pi], and sarcomere length  has been suggested to decrease Ca2+ sensitivity, mainly during early fatigue [i] is thought to cause a decrease in Ca2+ release from the sarcoplasmic reticulum (SR). This occurs because Pi can migrate into the SR and when [Pi] is high enough in relation to free [Ca2+] in the SR, a precipitate of Ca-Pi will result, which decreases the amount of Ca2+ available to be released m. It was also hypothesized that the fibers would fully recover force at high frequencies with persistent reduced force at low frequencies (low-frequency fatigue). Considering that most factors that depress Ca2+ sensitivity will recover within minutes [2+]m.Considering that potentiation due to RLC phosphorylation should occur early in a series of repeated submaximal contractions  and thatperature , it was  minutes , 20, it Healthy, adult female C57bl/6J  mice were used for this study. All mice were given standard care. Mice were killed by rapid cervical disarticulation and the whole flexor digitorum brevis (FDB) muscle was dissected to isolate single muscle fibers (see below).2, 0.5 MgCl2, 0.4 NaH2PO4, 24.0 NaHCO3, 0.1 EDTA, 5.5 glucose, and 0.2% fetal calf serum. The solution was bubbled with 5% CO2–95% O2 to keep pH at 7.4. The temperature of the Tyrode solution in the tissue bath was kept at 32 °C throughout the procedures. Indo-1 , a [Ca2+]m indicator, and 5 mM caffeine were also used in the experiments m indicator (< 100 μM), Indo-1, which was first dissolved in 150 mM KCl and 10 mM HEPES. Injection was followed by rest for 20–30 min to let the Indo-1 diffuse throughout the fiber. The fiber diameter was recorded as the average measured at both ends of the fiber. Diameter was used to calculate cross-sectional area, assuming a circular fiber. Excitation of Indo-1 was achieved by Xenon light source, passing through a monochromator to selectively illuminate the fiber at 360 nm. Emitted light was filtered and quantified at 405 ± 5 and 495 ± 5 nm with dual photomultiplier tubes . A FeliX32 Analysis Module  was used for data collection (sampling rate of 500 Hz) and for analysis.The single fiber was set to optimal length, determined with sequential 100 Hz stimulation for 350 ms, and was lengthened or shortened between contractions until the greatest isometric force was obtained. Each fiber was then microinjected with a [Ca2+]m, each fiber (n = 12) was stimulated at increasing frequencies  for 350 ms at 1-min intervals. A 5-min recovery period was allowed following the last contraction before the intermittent stimulation was initiated. In some cases, a longer rest interval was allowed because it was noticed that active force for 50 Hz stimulation was depressed relative to the value obtained during the above contractions. This extra rest (up to 30 min) was not sufficient to restore active force. An additional three fibers were tested with prolonged rest (5–10 min) between contractions at a reduced number of frequencies . This was done to evaluate the impact of potentiation or fatigue affecting the control force-[Ca2+]m relationship.To establish the control relationship between force and m.Intermittent contractions were elicited at 2-s intervals at a frequency of either 40 Hz  were superfused for 2 min with Tyrode solution containing 5 mM of caffeine, then stimulated at 200 Hz to assure maximal activation. After seeing four consecutive fibers producing similar force as when stimulated at 200 Hz at 37 min, the use of caffeine was stopped.Recovery started immediately after the 200 Hz contraction. At 5, 15, 25, and 35 min of recovery, the fiber was stimulated at three increasing frequencies , each for 350 ms at 1-min intervals to allow for the periodic assessment of the force-, ([Ca2+]m) was determined by measuring the ratio (R) of fluorescence at the two emission wavelengths (405 and 495 nm) after background subtraction [2+]m:Free myoplasmic [Catraction . The rattraction  to calcuKD is the apparent dissociation constant of Indo-1 [Rmin is the ratio at very low [Ca2+]m (obtained by injecting 0.5 M EGTA into the fibers to get resting [Ca2+]m); Rmax is the ratio at saturating [Ca2+]m ; and ß is estimated  was one of the constants yielded by fitting the data to Eq. 50 was obtained at the beginning and end of the intermittent contractions, and at each measurement time during recovery. This estimate used the same Hill coefficient as the control force-[Ca2+] relationship, and Ca50 and Fmax were adjusted to fit through the available data.m as illustrated in Fig. 2+]m. Fitting these data to Eq. 50 of 0.304 μM. When we compared this control force-[Ca2+]m curve to the first contraction of the intermittent stimulation protocol that followed, we noted that the first contraction occurred to the right of this control curve and at lower [Ca2+]m. This observation could be a result of potentiation of the original force-frequency data, and/or persistent fatigue resulting from completion of these contractions.The force-frequency data obtained during the first part of the standard protocol gave a sigmoidal relationship between active force and [Can = 3) were completed to see if the control force-[Ca2+]m relationship reflected fatigue and/or activity-dependent potentiation, explaining the difference in apparent Ca50. Fewer contractions at longer (5–10 min) intervals were obtained . When this was done, the first contraction of the intermittent contractions was closer to or superimposed on the control force-[Ca2+]m relationship so we concluded that the control force-[Ca2+]m relationship, obtained with 1-min intervals in the original fibers, reflected activity-dependent potentiation. For this reason, further comparisons during and following the intermittent contractions are between the apparent Ca50 for the first contraction of the intermittent contractions. This was assumed to represent the non-potentiated condition. The Ca50 for this contraction was obtained using the same maximum, minimum (fixed at zero), and slope for the control Hill equation. Ca50 was adjusted until the curve went through the value for the first contraction. The first contraction was consistently weaker than the control contraction at the same frequency. We attribute this to long-lasting fatigue resulting from the contractions used to establish the control force-[Ca2+]m relationship.Additional experiments (It took a fiber on average 77 ± 34 (mean ± SD) contractions to decrease force to 40% of initial force.During the time course of intermittent stimulation, expressed as percent of total time, an initial decrease in active force was observed (phase 1), followed by an increase in active force until about 30% into the duration phase 2). Following this, there was a slow decline, which usually was not sufficient to reach the 40% force target within 2 min. This target was achieved shortly after changing the frequency of contractions to one per second. The initial down then up staircase potentiation is shown in Fig. . Followi2+]m on an expanded time scale at different time points throughout the period of intermittent stimulation.Following this peak, force decreased in two phases to 40% of initial force . Raw data for one fiber during the fatigue protocol is shown in Fig. 2+ sensitivity. Accelerated relaxation is assumed to result in less summation of force for a given [Ca2+]m, reflecting a decreased Ca2+ sensitivity. Figure The fusion index (FUI) was calculated at 10% time increments throughout the time course of intermittent stimulation, to identify a potential mechanism for change in Ca2+]m and force appeared to change in a “P”-shaped pattern . From here, force increased first without a change in [Ca2+]m then with a corresponding decrease in [Ca2+]m (phase 2). Following the potentiation, force decreased, first in parallel with the initial force-[Ca2+]m relationship (phase 3), then without further change in [Ca2+]m (phase 4). Considering the small change in [Ca2+]m during phase 3, we evaluated the change to see if it reached statistical significance. A paired t test confirmed that this small change in [Ca2+]m from 3.722E-7 ± 5.5E-8M at 30% time to 3.31E-7 ± 4.7E-8M (mean ± SE) at 80% time was significant (p < 0.001). However, there was not a difference in average tetanic [Ca2+]m when comparing the start (3.2E-7M) and the end (3.2E-7M) of the intermittent contractions.From the first contraction to the end of the intermittent stimulation, the average [Caern Fig. . Initial2+]m curves have been superimposed on the data in Fig. 2+]m by the time the target force had been reached . This latter curve was produced using the same Hill coefficient as the initial curve, but changing Ca50 and the maximal value. This maximal value for active force was obtained by stimulation at 200 Hz as soon as the target force was reached. Half-maximal [Ca2+]m had increased to 3.45E-7M. Resting [Ca2+]m increased throughout the period of intermittent contractions, but recovered within seconds when stimulation was stopped , with post-hoc analysis showing a significant difference of Ca50 between start of fatigue and end of fatigue (p = 0.001). No significant difference was found when comparing Ca50 prefatigue to Ca50 at 5 and 15 min (p > 0.5). Values later in fatigue were not greater than the values observed at 5 min (data not shown).The Can = 3) at the beginning of the test and again at the end to see if a decrease in force or change in Ca2+ sensitivity occur as a consequence of shortening of sarcomeres which could be a consequence of tendon stretch or repositioning of the aluminum clips. There was only a 0.1 μm shortening in the three fibers from start of test to the end . This length change would not be expected to decrease active force in a mouse fiber in which optimal length of active force is achieved at a plateau from 2.4 to 2.8 μm m; and (4) only small changes in Ca50 were observed during the intermittent contractions. Each of these observations will be discussed below.The key observations of this study during fatigue and recovery from submaximal intermittent contractions are (1) [Ca2+]m relationship. A potential reason for this lower force is that the measure of force at 40 or 50 Hz in the force-frequency data was actually potentiated from previous stimulations , because the intervals between these contractions were only 1 min. Additional experiments confirmed this theory as the apparent force-[Ca2+]m relationship through the first contraction of the intermittent stimulation was very close to that of the force-frequency data when the force-frequency data were collected with intervals of at least 5 min. However, the active force for the first contraction of the intermittent stimulation was still suppressed relative to that obtained with the 5-min intervals due to lower [Ca2+]m. Apparently, eliciting the contractions for the control force-[Ca2+]m relationship already induces some long-lasting fatigue.It was observed that the fibers were seemingly already “fatigued” before the intermittent contraction protocol started. The first measurement of force was lower than the force measured at the same frequency in the prefatigue force-frequency data and the first contraction was to the right of the control force-[Ca2+]m , active force decreased  beyond the position of the initial contraction during the intermittent stimulation, as would have been expected during the staircase response consistently observed in our experiments. The change in active force during staircase is partly due to an increased Ca2+ sensitivity, so this mechanism would be consistent with RLC phosphorylation, but this change occurs relative to the already decreased Ca2+ sensitivity. This small relative change in Ca2+ sensitivity can only be explained by coexistence of a mechanism in the opposite direction m relationship  occurred with no further change in [Ca2+]m, indicating that the decline was due to a decrease in Ca2+ sensitivity, possibly in combination with a decrease in maximal force, which was evident at the end of the period of intermittent contractions  and compared to the Ca50 from prefatigue and the end of intermittent stimulation (postfatigue). Ca2+ sensitivity had decreased at the end of the intermittent contractions but had recovered such that Ca50 was not different from prefatigue values at both 5 and 15 min of recovery. During intense intermittent stimulation m was the primary mechanism for the persistent LFF following low-frequency contractions.After the fibers were fatigued, force and [Caery Fig. . Ca50 vantensity . The res2+]m, not decreased Ca2+ sensitivity [2+]m in LFF is due to disrupted excitation-contraction coupling [2+]m is likely due to impaired Ca2+ release from the SR as the amount of free [Ca2+]m in the SR should not be affected after a few minutes of recovery. Further research will be required to confirm this possibility.LFF was also observed by Westerblad et al.  and Chinsitivity , 40. Allsitivity  suggestecoupling . More sp2+ and/or that depletion of glycogen can cause decreased Ca2+ release [2+]m in the SR was not affected. This was also seen by Westerblad et al. [2+]m at 30, 50, and 200 Hz during the recovery period. In agreement with Westerblad et al. [2+]m observed throughout recovery indicates that decreased [Ca2+]m was the likely cause of low-frequency fatigue. This is consistent with the absence of change in Ca2+ sensitivity.Some researchers have indicated that increased ROS during repeated stimulation can inhibit the RyR, disrupting the release of Ca release , 42–44. d et al.  have sho2+]m differ during low-frequency compared with high-frequency intermittent stimulation reported previously. The resulting P-shaped change of force and [Ca2+]m has not been observed during high-frequency stimulation probably because low-frequency forces, which lie on the steepest portion of the force-[Ca2+]m relationship, are particularly sensitive to alterations in SR Ca2+ release and myofibrillar Ca2+ sensitivity that can increase as well as decrease force. Another novel observation was that force depression during the recovery period after fatigue induced by low-frequency stimulation was completely explained by impaired SR Ca2+ release, which is different from the consequences of high-frequency stimulation where impaired myofibrillar Ca2+ sensitivity also contributes to the persistent impairment of low-frequency force production. Thus, the mechanisms underlying fatigue and recovery of force generation are clearly task dependent.Novel findings of this study include identifying that the fatigue-induced changes in force and [Ca2+]m during intermittent submaximal contractions and subsequent recovery permits assessment of the primary mechanisms of fatigue. However, the underlying cause of the change in [Ca2+]m and Ca50 has not been determined. The potential impact of changes in Pi concentration and Ca2+ release represent best guesses at these mechanisms. The use of Indo-1 for detection of [Ca2+]m cannot detect quickly changing concentrations, but is suitable for resting and average concentrations during tetanic stimulation [The measurement of [Camulation .2+]m and Ca2+ sensitivity. Early in the intermittent contractions, the reduced force is due to decreased Ca2+ sensitivity. This is followed by an increased Ca2+ sensitivity while force potentiates above the original force, creating the loop part of the “P” force-[Ca2+]m pattern. During late fatigue, the decline in force is first due to decreased [Ca2+]m, changing to a decreased Ca2+ sensitivity, both of which create the shaft part of the “P” pattern. During recovery, the persistent depression of low-frequency force was completely explained by decreased [Ca2+]m. Clearly, there are different patterns of change in the relationship between force and Ca2+ during intermittent low-frequency contractions than have been reported for high-frequency contractions.In conclusion, results suggest that the mechanism of fatigue during submaximal intermittent contractions is a combination of changes to both [Ca


A 21-year-old male patient with no history of systemic disease or drug use presented to our clinic with redness and pain in the right eye. Best corrected visual acuity was 20/20 in both eyes. Inflamed pinguecula was observed on slit-lamp examination and the patient was prescribed ophthalmic nepafenac eye drops. After instilling the drops that day and the next day, the patient presented again due to pruritus and rash. Upon consultation with the dermatology department, the patient was diagnosed with drug-induced allergic urticaria and the nepafenac drops were discontinued. Although urticaria has been reported as a side effect after systemic non-steroidal anti-inflammatory drug (NSAID) use, such a reaction has not been reported with an ophthalmic NSAID and ours is the first reported case of urticaria following ophthalmic nepafenac use. This unique case highlights the fact that ophthalmologists must also keep urticaria in mind as a potential side effect when prescribing this drug. Ophthalmic NSAIDs currently in use include nepafenac, ketorolac tromethamine, diclofenac sodium, bromfenac and flurbiprofen. A study performed in rabbit eyes demonstrated the distribution of ophthalmic nepafenac in the cornea, aqueous humor, iris, ciliary body and choroid.10 ocular tissue hemorrhage,7 blurred vision, photophobia, posterior capsule opacity, foreign body sensation, dry eye and increased intraocular pressure.11 Adverse effects involving the pulmonary, gastrointestinal, dermatologic, renal, cardiovascular, hematologic, pulmonary and central nervous systems have been reported after topical, intramuscular and oral administration.19 Here we present urticaria as a previously unreported adverse effect of an ophthalmic NSAID.Adverse effects of ophthalmic NSAIDs include corneal melting,A 21-year-old male patient presented to our clinic with pain and redness in his right eye. On physical examination, visual acuity using Snellen chart was 20/20 in both eyes and intraocular pressures were 14 and 15 mmHg in the right and left eyes, respectively. On slit-lamp biomicroscopic examination, minimally inflamed pinguecula was noted on the nasal conjunctiva of the right eye. No pathology was observed in the left eye except pinguecula . Fundus 20NSAIDs act by inhibiting the enzyme cyclooxygenase (COX), thus reducing the synthesis of prostaglandin, prostacyclin and leukotriene from arachidonic acid. There are two forms of COX. COX-1 is generally found in all tissues and plays a protective role by regulating the action of prostaglandins. COX-2 increases inflammation by stimulating immune system cells and other tissues in the presence of various stimuli such as mitogens, inflammatory cytokines and tumor promoters.in vivo study in humans showed nepafenac had a significantly shorter time to peak anterior chamber concentration after instillation on the cornea, followed by amfenac, ketorolac and bromfenac.21 Ophthalmic nepafenac takes effect approximately 15 minutes after topical application and lasts more than 8 hours.22 Quantitative plasma concentrations of nepafenac and amfenac were measured in subjects 2-3 hours after ocular administration and mean steady-state C-max values of the drugs were 0.310±0.104 ng/mL and 0.422±0.121 ng/mL, respectively. Ophthalmic diclofenac has been associated with corneal melting in studies of the ophthalmic side effects of topical NSAIDs.7 In another study, topical ketorolac and bromfenac were associated with severe corneal damage and the authors suggested that patients with corneal damage should be asked about their use of these agents.9 Topical nepafenac has also been associated with corneal melting.10 Ophthalmic NSAIDs may prolong bleeding time by impairing platelet aggregation, thus leading to hemorrhage in ocular tissues.7 Therefore, caution is warranted when using ophthalmic NSAIDs long-term in patients using systemic NSAIDs, patients who smoke or use alcohol and in elderly and pediatric populations. In a study of the ocular side effects of nepafenac, ocular adverse events that occurred at rates of at least 1% included blurred vision, photophobia, posterior capsular opacity, foreign body sensation, dry eye and increased intraocular pressure.11Ophthalmic NSAIDs currently in use include nepafenac, ketorolac tromethamine, diclofenac sodium, bromfenac and flurbiprofen. The chemical designation of nepafenac is 2-amino-3-benzoylbenzeneacetamine and it is available as a 0.1% suspension. Ophthalmic nepafenac is the only prodrug among the NSAIDs. It is deaminated to form amfenac, a potent COX inhibitor. Ophthalmic nepafenac targets the anterior segment and intraocular vascular tissues. An 15 Another patient with no history of gastric ulcer developed gastric ulcer perforation four days after starting intramuscular ketorolac (NSAID) treatment for traumatic humerus and femur fracture.16 The systemic side effects of oral NSAIDs on the gastrointestinal, renal, cardiovascular, hematological, pulmonary and central nervous systems have been demonstrated in various studies.20 Dermatologic side effects include urticaria, morbilliform and vesiculobullous eruptions, exfoliative erythroderma, erythema multiforme, Steven Johnson syndrome and toxic epidermal necrosis.22Urticaria occurs as the result of mediator release from mast cells or basophils after contact with a triggering stimulus. These mediators induce vasodilation and transudation from small vessels, which causes the development of the characteristic erythematous, edematous, itchy papules and plaques. Many factors are implicated in the etiology of urticaria. The main etiological causes of acute urticaria are drugs, food and infections. It is usually possible to determine the etiology based on only a detailed history. Nearly all drugs can cause urticaria but the most common are antimicrobials , analgesics and antiinflammatory drugs , angiotensin converting enzyme (ACE) inhibitors and blood products.24 In a study conducted in rabbits, it was determined that ocular instillation of 0.5% (50 µL) diclofenac resulted in a peak plasma concentration of 185 ng/mL after 15 minutes at the earliest.25 In addition, it has been shown in rabbits that 7-10% of ophthalmic flurbiprofen enters the ocular circulation, while 74% passed to the systemic circulation.26 Urticaria is a known adverse effect of systemic NSAID use and we believe that our patient developed it after the ophthalmic NSAID entered the systemic circulation via the conjunctival vessels and nasolacrimal duct. Although there are previous reports of allergic urticaria after oral NSAID use,24 our case is novel as the first reported case in the literature of allergic urticaria as an adverse event after ophthalmic NSAID use.Adverse effects have also been reported after using topical and intramuscular NSAIDs. In one of these reports, a patient with asthma history experienced an asthma attack after using piroxicam topical gel (NSAID) for knee pain.


Strikingly, talazoparib and APE1 inhibitor III demonstrated critical antileukemic efficacy in selected MDS/CMML and AML cell samples. Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in MDS/CMML and AML cells. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in MDS/CMML and AML cells. In summary, talazoparib and APE1 inhibitor III demonstrated substantial antileukemic efficacy as single agents, in combination with decitabine, and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, in primary CD34+ MDS/CMML cell samples ( Survival of patients belonging to the unfavorable risk groups of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) is poor after standard antileukemic therapy ,2,3,4,5.Recent advances in the understanding of the biology of MDS/CMML and AML led to the development of novel targeted and untargeted agents  ,18,19,20Talazoparib is a potent inhibitor of PARP1, which is involved in SSB-R and BER . PARP1 sThe APE1 inhibitor III inhibits APE1 , which iFinally, 5-aza-2´-deoxycytidine (decitabine) is a potent DNMTi, which is used for the treatment of MDS/CMML ,7,8 and PARP1/APE1 mRNA expression, γH2AX foci levels, chromosomal aberrations, and gene mutations with cell survival.In this study, we analyzed the antileukemic efficacy of talazoparib and APE1 inhibitor III in MDS/CMML and AML cells with defects in DSB repair. The cytotoxic efficacy of talazoparib and APE1 inhibitor III were tested alone, combined with decitabine, and combined with each other in primary CD34+ MDS/CMML cells and in primary CD34+ or CD34− AML cells in comparison to healthy CD34+ donor cells. We further analyzed the correlation of distinct parameters and biomarkers including cell proliferation, n = 8; 4 MDS and 4 CMML samples) and in CD34+ or CD34− AML cells (n = 18) in comparison to healthy CD34+ donor cells (n = 8)  cytotoxic efficacy of talazoparib in 2 MDS/CMML  and 3 AML cell samples  (7 nM ± 2 (mean IC50 ± standard error of mean)) as compared to the 8 healthy donor cell samples (16 nM ± 2) (p = 0.059) in 1 MDS (MDS#2) and 5 AML cell samples  (603 nM ± 71) as compared to the cytotoxic efficacy in 8 healthy donor cell samples (1041 nM ± 149)  . The com nM ± 2) A. The ’rM ± 149) B. The ’rThe cell proliferation rate of MDS/CMML and AML cells might correlate with the cytotoxic efficacy of talazoparib and APE1 inhibitor III, respectively. Therefore, growth curves of untreated MDS/CMML and AML cells were correlated with the corresponding surviving fractions of talazoparib and APE1 inhibitor III treated MDS/CMML and AML cells C,D. HoweThe cytotoxic efficacies of (I) decitabine ± talazoparib, (II) decitabine ± APE1 inhibitor III, and (III) talazoparib ± APE1 inhibitor III were analyzed in CD34+ MDS/CMML cells and in CD34+ or CD34− AML cells . SubtoxiPARP1 and APE1 mRNA expression might correlate with the cytotoxic efficacy of talazoparib and APE1 inhibitor III in MDS/CMML and AML cells and explain why some of the patients did not respond to the drugs. Therefore, PARP1 and APE1 mRNA expression levels were analyzed in MDS/CMML and AML cell samples. However, the PARP1 and APE1 mRNA expression levels did not differ significantly between ‘responders’ and ‘non-responders’  were analyzed for their potential correlation with the cytotoxic efficacy of talazoparib and APE1 inhibitor III in MDS/CMML and AML cells. Therefore, γH2AX foci levels were quantified in MDS/CMML and AML cell samples. However, the number of γH2AX foci did not significantly differ between ‘responders’ and ‘non-responders’ , 50% of NRAS mutated AML samples , and 67% of U2AF1 mutated MDS/AML samples  were sensitive towards talazoparib and APE1 inhibitor III, respectively. On the other hand, the AML sample (AML#12) most insensitive towards talazoparib (IC50 505 nM) was TP53-mutated and harbored a complex karyotype. Further, the AML sample (AML#9) most insensitive towards APE1 inhibitor III (IC50 5781 nM) was DNMT3A-, FLT3-TKD-, IDH1-, NPM1-, NRAS-mutated and harbored a normal karyotype. Distinct chromosomal aberrations and gene mutations might correlate with the cytotoxic efficacy of talazoparib and APE1 inhibitor III in MDS/CMML and AML cells. Therefore, a comprehensive analysis of chromosomal aberrations and gene mutations was performed in MDS/CMML and AML cell samples . CytogenIn the present study, the cytotoxic efficacy of talazoparib and APE1 inhibitor III was investigated using several distinct approaches in CD34+ MDS/CMML cells and in CD34+ or CD34− AML cells in comparison to healthy CD34+ donor cells. In addition, potential predictive markers were analyzed and correlated with the cytotoxic efficacy of talazoparib and APE1 inhibitor III.Talazoparib demonstrated critical antileukemic efficacy as a single-agent and in combination with decitabine in several MDS/CMML and AML cell samples, which might be mediated by the formation of cytotoxic PARP1-DNA complexes , DNMT1-D50 values as compared to talazoparib. Our observations are in accordance with the more advanced development of PARP inhibitors  as compared to APE1 inhibitors . Despite the inferior potency of APE1 inhibitor III, subtoxic doses increased the antileukemic efficacy of decitabine and talazoparib in about 78% and 68% of MDS/CMML/AML cell samples, respectively. As BER is presumed to be involved in the repair of decitabine-induced DNA lesions [Furthermore, APE1 inhibitor III demonstrated antileukemic efficacy in several MDS/CMML and AML cell samples. The ‘responder’ rate of 25% in MDS/CMML/AML cell samples treated with APE1 inhibitor III was similar to the ‘responder’ rate of 19% in MDS/CMML/AML cell samples treated with talazoparib. However, APE1 inhibitor III exhibited about 30-fold less potent IC lesions , we hypo lesions .γH2AX foci levels, chromosomal aberrations, and gene mutations were analyzed and correlated with cell survival of MDS/CMML and AML cells after treatment with talazoparib and APE1 inhibitor III, respectively. Cell proliferation, PARP1/APE1 mRNA expression, and γH2AX foci levels demonstrated no consistent correlation with the cytotoxic efficacy of talazoparib and APE1 inhibitor III in MDS/CMML and AML cells. Furthermore, no specific chromosomal aberrations with a predictive impact were detected. However, 100% of the talazoparib ‘responders’ and 83% of the APE1 inhibitor III ‘responders’ displayed a normal karyotype. Finally, gene mutation analysis suggested that KMT2A-PTD, NRAS, and U2AF1 mutations might be associated with a sensitivity rate of 50% or more towards talazoparib and APE1 inhibitor III, respectively.Markers predicting the cytotoxic efficacy of talazoparib and APE1 inhibitor III in MDS/CMML and AML cells are, so far, largely unknown. Therefore, several markers including cell proliferation, PARP1/APE1 mRNA expression, BRCA1/2, loss of 53BP1, loss of Shieldin factors), and (IV) stabilization of stalled forks   increased drug efflux , (II) decreased trapping of PARP1 on DNA , (III) restoration of homologous recombination . FurtheThis study was approved by the Ethics Committee II of the Medical Faculty of Mannheim at Heidelberg University (2018-566N-MA). Written, informed consent was obtained from all participants. Heparinized bone marrow (BM) samples were collected from 8 healthy donors , 8 MDS/CMML patients , and 18 AML patients  prior to antileukemic therapy .2 atmosphere at 37 °C in StemSpan medium  supplemented with StemSpan myeloid expansion supplement  (Stemcell Technologies Germany GmbH) and 1% penicillin/streptomycin.Mononuclear cells were separated from BM samples by Ficoll density gradient centrifugation. CD34+ cells were enriched using CD34 microbeads and cell separation columns . Healthy CD34+ donor cells, CD34+ MDS/CMML cells, and CD34+ or CD34− AML cells were grown in a humidified 5% COMDS/CMML and AML BM samples were diagnosed according to standard procedures using cytomorphology , flow cytometry, G-banding cytogenetics, fluorescence in situ hybridization (FISH), and molecular mutation analysis ,49,50.Cell proliferation of untreated MDS/CMML and AML cells was determined by trypan blue exclusion assay at days 1, 2, and 3 .γH2AX was performed in untreated healthy donor cells, MDS/CMML cells, and AML cells using a mouse monoclonal anti-γH2AX antibody  and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody  as previously described [Immunofluorescence staining of escribed ,53.PARP1 and APE1 were analyzed in healthy donor cells, MDS/CMML cells, and AML cells. For quantitative real-time PCR (qRT-PCR), the QuantiFast SYBR Green PCR Kit  with commercial bioinformatically validated primer sets for PARP1  (Qiagen) and APE1  (Qiagen) was used according to manufacturer instructions. Normalization validation was performed with a primer set for the reference gene GUSB  (Qiagen). Samples were analyzed in triplicate using the LightCycler 480 II system  equipped with 96-well plates with a total well volume of 25 µL containing primer mix (2.5 µL), SYBR Green Master Mix (12.5 µL), 20 ng template cDNA (2 µL), and PCR-grade water (8 µL). qRT-PCR was performed at thermal cycling conditions of 95 °C for 5 min, 95 °C for 10 s, and 60 °C for 30 s (×40 cycles).The mRNA expressions of Stock solutions of 50 mM talazoparib III , 5 mM APE1 inhibitor III , and 50 mM decitabine  were prepared by dissolving the agents in dimethylsulfoxide (DMSO) (Merck).50 designated the drug concentration capable of killing 50% of the cells and was calculated with GraphPad Prism 5 software  by linear regression. MDS/CMML and AML patient samples were classified as ‘responders’ or ‘non-responders’ using a cutoff value of mean IC50 of healthy controls minus standard error of mean. Experiments were performed once owing to the limited availability of healthy CD34+ donor cells, CD34+ MDS/CMML cells, and CD34+ or CD34− AML cells. DMSO-treated cells were used as control.The CellTiter-Glo luminescent cell viability assay  was used for the assessment of cell survival of healthy donor cells, MDS/CMML cells, and AML cells according to the manufacturer’s instructions. Cryopreserved CD34+ MDS/CMML cells and CD34+ or CD34− AML cells were thawed and expanded in culture for 3 d followed by 3 d of daily drug exposure. The ICp values < 0.05 were considered as statistically significant.All statistical calculations were done with SAS software, release 9.4 . For quantitative variables, mean values and standard errors were calculated. Categorical factors are presented with absolute and relative frequencies. In order to compare more than two groups, Kruskal–Wallis tests were performed. For pairwise comparisons, Wilcoxon two-sample tests were used. Test results with PARP1 and APE1 expressions, relative quantification was used according the ΔΔCT method. Normalization of target genes was performed using the arithmetic mean Ct of the nonregulated housekeeping gene GUSB from 6 healthy donor samples.For analysis of Our study performed on primary MDS/CMML and AML patient cells provides important preclinical data for a potential clinical use of talazoparib and APE1 inhibitor III alone and in combination with decitabine. In a next step, the in vivo antileukemic activities of talazoparib and APE1 inhibitor III need to be evaluated in clinical studies. Currently, talazoparib is tested as a single agent in advanced hematologic neoplasias (NCT01399840)  and in c


Objective: In aging population, postmenopausal osteoporosis and decline of musculoskeletal function, referred to as “frailty syndrome” lead to loss of bone and muscle, causing falls, and fall-related injuries. To limit the impact of this portentous duo, simultaneous treatment of both is needed. Urocortin (UCN) has been reported to improve osteoporotic bone properties while its effect on muscle has not been addressed yet.Design and Methods: We aimed to investigate the effect of urocortin in vivo on skeletal muscle structure in osteopenic rats. Sixty Sprague-Dawley rats were divided into five groups: four were ovariectomized (OVX) and one underwent sham operation (SHAM). One ovariectomized group was left untreated (OVX), while one was treated with urocortin s.c. in 3 μg/kg body weight (bw) (OVX+UCN low), one with 30 μg/kg (OVX+UCN high), while one group was treated with estradiol orally (OVX+E: 0.2 mg/kg bw), each for 35 days. Mm. gastrocnemius, longissimus, and soleus were isolated and capillary density as well as diameters of type I and II fibers were measured. In addition, we examined the effect of UCN on tibia using biomechanical, micro-CT and ashing analysis and investigated the blood serum.Results: We demonstrated a positive effect of UCN on M. soleus, in which fiber diameter was positively influenced. The biomechanical and structural parameters of bone were not changed in UCN treated rats. The higher cholesterol, glucose and triglyceride levels in the “UCN high” group raise concern about this treatment.Conclusions: Our results portray urocortin as a substance that can be assessed for future therapeutic treatments of estrogen deficiency.New and Noteworthy: Urocortin has a positive effect on M. soleus (diameter). Urocortin raises serum cholesterol and triglyceride levels. Bone tissue was not affected by UCN. Due to the high prevalence of postmenopausal osteoporosis , 2, a stHence, innovative therapeutic strategies against muscle loss need to address both, bone and muscular systems.in vitro (in vivo (Urocortin I (UCN), a peptide consisting of 40 amino acids , and itsin vitro –28 and i and M. soleus (mainly slow fiber types) as lower leg agonists were chosen as surrogates for different fiber types, while M. longissimus (with mainly fast fiber types) represents a holding muscle. We sought to investigate the changes in muscle fibers and capillarization as parameters of nutrient supply  were kept at 22°C with 55% relative humidity in Makrolon IV®-Cages. One acclimatization week was included prior to the beginning of the experiment, which took place in conformance with the ethical standards of animal care. The experimental design was approved by the local institutional animal care and use committee .At an age of 13 weeks, the rats underwent bilateral ovariectomy (OVX) or sham surgery (SHAM) as described elsewhere . OperatiBased on previous studies, around 8 weeks after OVX, the rats were expected to develop osteoporosis with concomitant hormone withdrawal , 52, 53.2-anesthetized and decapitated.The OVX+Estradiol group was supplied via food (10 mg/kg food) with 0.2 mg/kg bw estradiol-17-benzoate  per day for 35 days. All rats received a soy-free diet  during the experiment. Food intake and body weight were recorded once weekly and are already published , 40. In Mm. gastrocnemius and soleus were weighed and transversely cut in half across the whole muscle. A piece of M. longissimus (1 × 3 cm) was extracted. After that, the muscles were snap-frozen in liquid nitrogen and stored at −80°C. Afterwards, 12 μm frozen sections were cut orthogonally with a cryotome .Rats were sacrificed after 35-day treatments. Uterus, heart, liver, kidneys, spleen and visceral adipose tissue were extracted and weighed. M. gastrocnemius and M-longissimus (type I)  staining was carried out as described elsewhere . Three d(type I)  52). In. InM. gae type I . TherefoFor the relationship of fiber to capillaries, periodic acid-Schiff (PAS) staining was performed, following the method of Andersen , 57. TwoThe selection of image areas as well as their analyses have been performed by one individual who was blinded with respect to the treatment groups.2 × 6H2O, 1 mM EGTA, pH 7.5). Afterwards, they were assessed with a spectrophotometer . The assay was performed within 2 h following the tissue homogenization. The lactate dehydrogenase (LDH) activity was indirectly measured by recording the reduction in absorption at 340 nm, which demonstrates the oxidation of NADH to NAD+  activity was measured at 412 nm with Triton X-100 (0.1%) for the liberation of all CS proteins according to Faloona and Srere , whereasTibia biomechanics was assessed using a Zwick device  as previously described . Four sc2) as well as bone volume fraction (BV/TV) were assessed at the metaphysis. Therefore, two measurements next to the growth plate were performed (3 and 5 mm), and the area in between was cut out. The volume and density of cortical, trabecular and total bone were measured in this field. After turning 90°, the cross-section was used to measure the distal cortical bone area (Ct.Ar), endosteal area (E. Ar), and total area (T. Ar.) of the bone. Thereafter, the cortical bone was cut off, and trabecular structure was analyzed. Trabecular thickness (Tb.Th), number of trabecular nodes (N.Nd), trabecular number (Tb.N), mean trabecular junctions at one node (Tb.N/Nd), and trabecular separation (Tb.Sp) were measured  with 72 kVp, 90 μA, and 1,600 ms exposure time and 360° rotation, resulting in 0.0029 mm pixel size. Five hydroxyapatite blocks with defined mineral densities were scanned to transform gray scale values into bone mineral density (mg/cm3). 3D OsteoAnalyze, which was developed for our laboratory, was used to determine bone parameters according to American Society for Bone and Mineral Research (ASBMR) criteria , 68–70. measured , 71.The tibia was ashed at 750°C for 1 h. Afterwards, calcium content was quantified with an atomic absorption spectrometer . Orthophosphate content was assessed by the colorimetric method  , 72. TheSerum analyses were conducted at the Department of Clinical Chemistry, University of Goettingen according to the manufacturers' instructions .After collecting 5 ml of whole blood from the rats in the end of the experiment, it was centrifuged at 3,000 × g for 10 min. Serum was stored at −20°C. Creatine kinase (CK), Alanine transaminase (ALT), triglycerides, uric acid, Aspartate aminotransferase (AST/GOT), Cholesterol, glucose, and HDL were measured with the ARCHITECT c System and AEROSET System .p < 0.05, F test), the Tukey-Kramer post hoc test (α = 0.05) was performed to estimate the differences between individual means . One-way ANOVA  was conducted to reveal the impact of the treatments on variables. For variables that showed an overall statistically significant difference in group means  and OVX+Estradiol (324.3 ± 16.5 g) groups was significantly lower compared to the OVX (396.8 ± 18.9 g), OVX+UCN high (388.3 ± 43.7 g) and OVX+UCN low (375.2 ± 16.6 g) groups . Body w < 0.05; ]. ThoughM. soleus, OVX+UCN high caused significantly higher values compared to all other groups  treatments  compared to OVX+UCN low (55.3 μm) (In 55.3 μm) . Type II55.3 μm) .M. soleus, the mean diameter of fibers was significantly higher in OVX+UCN high (81.6 μm) compared to SHAM (67.0 μm), OVX (59.9 μm), and OVX+Estradiol (68.2 μm)  . Effect M. gastrocnemius  activity in e groups .M. gastrocnemius  activity, no differences could be obtained in ocnemius  as in M.gissimus . In M. sX groups .M. gastrocnemius  activity showed the highest levels in OVX compared to all other groups in ocnemius , whereasdetected .Enzyme activity in terms of citrate synthase was significantly enhanced by UCN high treatment, whereas UCN therapy did not lead to differences in LDH or CI activity.After detection of metabolic muscle alterations, we proceeded to assess bone parameters to learn whether the former lead to changes in the subjacent bone. While stiffness was not altered, yield load and maximum load were significantly diminished in the OVX+UCN low group compared to SHAM and OVX+Estradiol. In the OVX group, yield load and maximum load were lower than in the SHAM group .In the ashing analysis, the calcium content was significantly lower in OVX+UCN low group and OVX+UCN high than in SHAM , correspUCN treatment did not cause biomechanical changes or mineral differences in bone of OVX rats.As a next step we investigated the microstructure of tibiae. Notably, micro-CT analysis showed that all OVX groups, compared to SHAM, had reduced total bone mineral density . Lower tFinally, to assess the potential systemic impact of urocortin, investigation of serum parameters followed. Neither CK nor AST/GOT was significantly different among the groups , though To detect the impact of urocortin on musculoskeletal metabolism, we used an established osteoporotic rat model , 66. FivSuccessful ovariectomy was demonstrated by the significantly raised body weight and reduced uterus weight in all OVX groups, while estrogen treatment lead to similar body weight compared to SHAM rats and an increase in uterus weight; UCN treatments did not alter body weight or uterus weight .M. gastrocnemius, which functions as a dynamic muscle because it contains more fast-twitch type IIb fibers in rats than in humans. In the slow-twitch type I fibers and type IIb fibers as well, no differences in diameter after treatments with urocortin were detected. The M. soleus, as a holding muscle, mainly consists of slow and constantly contracting type I fibers , while in our study, 3 μg/kg  and 30 μg/kg  were applied. In the study of Hinkle et al.  than in M. gastrocnemius, in which the UCN did not inhibit muscle loss at any dose studied. In the glucocorticoid-induced muscle atrophy model, UCN inhibited muscle loss at all doses tested  inhibited casting-induced muscle mass loss, myocyte cross-sectional area loss and force loss  mice has been seen at a dose of 300 μg/kg (bw) after 2 weeks (compared to our 35 days), which was 10-fold higher than the highest dose that we injected . Hinkle The adverse effects of urocortin on the cardiovascular system, with raised values of cholesterol, HDL and triglycerides, cause solicitude. Particularly, the impact of our highest UCN concentration on cholesterol has not been described in the literature. However, the cardio-protective effect of UCN is well-known –82. Our Since, after entering blood circulation, the potential site of action for UCN are osteoblasts and osteoclasts as well as myocytes and immune cells , 86, we Regarding bone parameters, biomechanical evaluation revealed reduced yield load and maximum load in the UCN low group compared to SHAM and OVX+Estradiol group, similar to that in the OVX group. In the UCN high group, the biomechanical properties of bone were at the level of SHAM and Estradiol groups. Similar results were demonstrated in the inorganic mass in the ashing analysis, confirming the results of our group on rat femora and vertebrae, where UCN showed overall positive effects on bone parameters , 40. OneM. quadriceps femoris would above the femur, which was not examined in our study. Contradictory, M. longissimus did not show significant effects despite spine did.These results are, however, contradicted by the calcium levels measured in our study, where both UCN groups had the lowest levels. More precise evaluation of tibia with micro-CT did not confirm the harmful effects of low-dose urocortin treatment on trabecular or cortical bone, but it also did not confirm the positive effects that have been reported for high-dose urocortin treatment in spine and femur , 40. A pWe conclude that the effects of urocortin I seem to be fiber-type specific and that type I fibers benefit more from the treatment. Though postmenopausal muscle loss is a phenomenon known to affect fast-twitch muscle fibers more than slow-twitch fibers, Urocortin seems to be a promising substance for muscle tissue in estrogen-deficient organism.M. soleus compared to the SHAM, OVX, and OVX+Estradiol groups. The ratio of capillaries to muscle fibers was higher in M. soleus after UCN high treatment, whereas parameters of muscle damage in serum were not altered due to urocortin-treatment. Bone parameters were not improved significantly, whereas parameters of metabolism during urocortin treatment, including increased cholesterol and triglycerides, should be examined more deeply in future studies. Urocortin is a promising substance for further studies regarding skeletal muscle, but its optimal dose of application and potential harmful effects on healthy and diseased tissues need to be further characterized.The diameter of oxidative muscle fibers increased after UCN treatment in In our model, functional muscle tests like strength test, wire hang test or different activity tests were not implemented. Therefore, the assertions regarding functional muscle parameters, although indirectly obtained by muscle enzyme activity and structural parameters, are missing.There was no formal, blinded analysis of behavior: future studies should formally assess the effects of UCN treatment on home cage locomotor activity since effects on locomotor activity could affect muscle physiology.M. longissimus was not measured which is also a limiting factor in this study. The effect of UCN on skeletal muscle in the OVX rat is limited compared to denervated or casted mice since the latter leads to a well-defined muscle atrophy, while the former mimics the estrogen-deficit in postmenopausal women.Since this study does not include sarcopenic rats, the described effects just show the influence of UCN on skeletal muscle in an estrogen deficient osteoporotic rat model. Furthermore, the weight of Measured bone parameters are just static and do not imply analysis of osteoblast and osteoclast relation or bone turnover. Ultimate statements regarding the safety of UCN cannot be made until all possible side effects would be investigated in detail.The experimental design was approved by the local institutional animal care and use committee .DS, LG, TG, DH, MT, and MK conducted the experiments. DS, MT, SS, and MK interpreted the results. The final manuscript was drafted by DS, reviewed by SS and MK, and approved by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, the expression of voltage-gated Ca2+ (Cav) channels was determined by real-time RT-PCR and their significance for the cellular TTFields response defined by knock-down and pharmacological blockade. As a result, TTFields stimulated in a cell line-dependent manner a Cav1.2-mediated Ca2+ entry, G1 or S phase cell cycle arrest, breakdown of the inner mitochondrial membrane potential and DNA degradation, and/or decline of clonogenic survival suggesting a tumoricidal action of TTFields. Moreover, inhibition of Cav1.2 by benidipine aggravated in one glioblastoma line the TTFields effects suggesting that Cav1.2-triggered signaling contributes to cellular TTFields stress response. In conclusion, the present study identified Cav1.2 channels as TTFields target in the plasma membrane and provides the rationale to combine TTFields therapy with Ca2+ antagonists that are already in clinical use.Tumor treating fields (TTFields) represent a novel FDA-approved treatment modality for patients with newly diagnosed or recurrent glioblastoma multiforme. This therapy applies intermediate frequency alternating electric fields with low intensity to the tumor volume by the use of non-invasive transducer electrode arrays. Mechanistically, TTFields have been proposed to impair formation of the mitotic spindle apparatus and cytokinesis. In order to identify further potential molecular targets, here the effects of TTFields on Ca Tumor Treating Fields (TTFields) therapy, developed by the company NovoCure  is a new modality of anti-cancer therapy which has been FDA-approved for newly diagnosed and recurrent glioblastoma multiforme and is currently evaluated for several other tumor entities. TTFields apply alternating intermediate-frequency 200 kHz) sine wave-electric fields with low field strength 1–3 V/cm) to the tumor with the help of non-invasive ceramic transducer electrode arrays . TTFields were usually administered 20–24 h/day for several months i) and Ca2+-dependent ion channel activity in the plasma membrane in individual human glioblastoma cells with the help of a single cell TTFields applicator disclosing a TTFields-stimulated Ca2+ entry that involves Cav1.2 channels. In a second step, effects of TTFields on cellular DNA content, mitochondrial membrane potential (ΔΨm) and colony formation were analyzed in dependence on pharmacological Cav blockage with the help of a TTFields cell culture applicator in order to assess cell cycle distribution, asymmetric cell division, cell death, triggering of intrinsic apoptosis, and clonogenic survival.Our study placed emphasis on Cal fields . Likewis signals ,30,31,32view see ,34,35). coupling . Notablyma cells . Potentince BKCa ,39,40,41channels ,43,44 thSilencer® Select Negative Control No. 1 siRNA, #4390844, Ambion™, Thermo Fischer Scientific). Detached T98G and U251 cells /10% FCS medium, respectively) were added to 500 µL of pre-incubated (20 min at room temperature) Opti-MEM medium containing RNAiMAX lipofectamine  and siRNA .Human T98G  and U251  glioblastoma cells were grown in 10% fetal calf serum (FCS)-supplemented RPMI-1640 (T98G) or DMEM  medium. For CACNA1C knock-down, exponentially growing U251 and T98G cells were reversely transfected with a mixture of three Stealth siRNAs  specific for human CACNA1C  or with nt siRNA  were incubated with fura-2/AM  in RPMI-1640/10% FCS and DMEM/10% FCS medium, respectively. free[Ca2+]i was recorded at 37 °C during superfusion with Ca2+-containing NaCl solution , with Ca2+-free NaCl solution , or with Ca2+-containing NaCl solution further containing the L-, N-, T-type Ca2+ channel blocker benidipine or the L-type inhibitor nifedipine  before, during and after application of TTFields .Fluorescence measurements were performed at 37 °C using an inverted phase-contrast microscope . Fluorescence was evoked by a filter wheel -mediated alternative excitation at 340/26 or 387/11 nm . Excitation and emission light was deflected by a dichromic mirror  into the objective  and transmitted to the camera (Visitron Systems), respectively. Emitted fluorescence intensity was recorded at 587/35 nm (AHF). Excitation was controlled and data acquired by Metafluor computer software . The 340/380-nm fluorescence ratio was used as a measure of Currents of semi-confluent T98G and U251 cells were elicited by 33 voltage square pulses (700 ms each) delivered in 5 mV increments from 0 mV holding potential to voltages between −80 mV and +80 mV and recorded at 37 °C in cell-attached, voltage-clamp mode by an EPC-9 amplifier  using Pulse software (Heka) and an ITC-16 Interface .2+-containing NaCl solution (see above). Borosilicate glass pipettes  manufactured by a microprocessor-driven DMZ puller  filled with Ca2+-containing NaCl solution (see above) were used in combination with a STM electrical micromanipulator . Macroscopic cell-attached currents were analyzed by averaging the currents between 100 and 700 ms of each square pulse. In addition to macroscopic cell-attached currents unitary current transitions were characterized for single channel conductance and open probability (Po). The latter was estimated by subtracting the zero current  at a given clamp voltage from the averaged macroscopic current and by dividing the difference by the amplitude of the unitary current transition and by the apparent number of active channels.Clamp voltages refer to the intracellular face of the plasma membrane. Flow of positive charge out of the cells (or the counter flow of anions) is defined as positive current and depicted as upward deflection of the current tracings. Cells were superfused at 37 °C with Ca® RNA kit, Machery-Nagel, Düren, Germany) and reversely transcribed and CACNA1A, -1B, -1C, -1D, -1E, -1G, -1H, -1I, -1S as well as housekeeper β-actin (ACTB)-, pyruvate dehydrogenase beta (PDHB)-, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific fragments were amplified by the use of SYBR Green-based quantitative real-time PCR  in a Roche LightCycler®480 Instrument . mRNA abundances were normalized to the geometrical mean abundance of the three housekeepers. RNA of control, CACNA1C siRNA-, or nt siRNA-transfected U251 and T98G cells (48 h after transfection) was isolated (NucleoSpin1 (DNA degradation) and hyper-G (aneuploidy) populations, respectively. Data were analyzed with the FCS Express 3 software . Exponentially growing T98G and U251 cells were treated for 5–7 days with TTFields  in the presence of benidipine (0 or 1 µM in 0.1% DMSO), trypsinized, washed, and stained (30 min at room temperature) with propidium iodide  in RNase-containing phosphate-buffered saline (PBS) further containing 0.1% Na-citrate, 0.1% triton X-100, and PI (10 µg/mL). For cell cycle analysis, DNA amount was recorded by flow cytometry  with FL-3  emission wavelength. In addition, DNA content was monitored with FL-2 emission wavelength  for determination of sub-G2+-containing NaCl solution (see above) containing the ΔΨm-specific dye tetramethylrhodamine ethyl ester perchlorate . TMRE-specific fluorescence was measured by flow cytometry with FL-2 (585/42 nm) emission wavelength.TTFields-treated  and benidipine-co-treated (0 or 1 µM in 0.1% DMSO) T98G and U251 cells were trypsinized, washed, and incubated for 30 min at room temperature in CaTo test for clonogenic survival, T98G and U251 were pre-plated (600 cells/well in 12-well plates), co-incubated with benidipine (0 or 3 µM in 0.3% DMSO) and subjected to TTFields  for 5–7 days and further grown for 10–14 days in the continuous presence of benidipine. Thereafter, clusters of ≥ 50 cells were defined as colonies and counted manually after fixation and Coomassie staining. The plating efficiency was defined by dividing the number of colonies by the number of plated cells. Survival fractions were calculated by dividing the plating efficiency of the TTFields-treated cells by those of the TTFields-untreated vehicle controls.p) of statistical significance was estimated with two-tailed t-test or Welch-corrected two-tailed t-test where appropriate. p values of p ≤ 0.05 (2 samples) or np ≤ 0.05 (> 2 samples) was assumed to be significantly different with n = number of pair wise comparisons in multiple testing (Bonferroni correction).Data are given as means ± standard error (SE). Probability  by ratiometric fura-2 Ca2+ imaging. As a result, acute application of TTFields to U251 and T98G glioblastoma cells induced a long-lasting increase in free[Ca2+]i in an electric field intensity (0.25–2.5 V/cm)-dependent manner  by continuous cell-attached patch-clamp recording with physiological extracellular NaCl solution in bath and pipette  of around −60 mV in T98G cells (+-containing) NaCl pipette and bath solution, the (negative) physiological membrane potential remains unaltered and applies on top of the clamp voltage across the recorded membrane. Thus, the actual reversal potentials of the I/V curves in + equilibrium potential indicating its K+ selectivity.Remarkably, the macroscopic currents in 8G cells F, top an8G cells F, bottomo) of this high conductance K+ channel  could also not be resolved. The same holds true for IKCa -mediated outward current transitions. Nevertheless, our observations suggest that the observed TTFields-induced increase in free[Ca2+]i is functionally relevant and sufficient to activate BK K+ channels.The I/V curves in free[Ca2+]i rise in further fura-2 experiments by analyzing its dependence on extracellular Ca2+ and its sensitivity to the Ca2+ channel inhibitors benidipine and nifedipine . In both glioblastoma lines, decrease of extracellular Ca2+ by superfusion with Ca2+-free, EGTA-chelated NaCl solution  ab51 cells B,D,E. Nomulation C. Togeth2+ channels in our two human glioblastoma cell lines and in glioma resection specimens by RT-PCR and by querying the TCGA low grade glioma and provisional glioblastoma data bases, respectively. Among all tested mRNAs, the mRNA of the L-type channel CACNA1C (Cav1.2) was most abundant in T98G and U251 cells  and benidipine (0 or 1 µM) and determined the cellular DNA content by propidium iodide staining in flow cytometry thereafter. In T98G, TTFields showed a tendency to decrease G1 population and significantly increased S and decreased G2 populations  was quantified as a measure of chromosome aneuploidy. In T98G, benidipine tended to elevate and TTFields increased hyper-G population, and both effects seemed to be additive (m) by TMRE staining in flow cytometry in T98G and U251 cells treated with TTFields (0 or 1 V/cm) and benidipine (0 or 1 µM) using time schedules identical to those applied for cell cycle analysis. In T98G, TTFields showed a trend to induce ∆Ψm dissipation as indicated by low TMRE staining while benidipine alone had no effect. In combination, however, TTFields and benidipine evoked a doubling of cell population with dissipated ∆Ψm suggesting their synergistic action , effects that were not additive in combined treatment  and most probably further Cav channels and which was completely suppressed by Ca2+ channel inhibitor benidipine. Cell line-dependently, TTFields induce S (T98G) or G1 cell cycle arrest (U251), aneuploidy, and DNA degradation (T98G), triggered intrinsic apoptosis (U251), and decreased clonogenic survival (T98G). Importantly, in T98G cells which exhibited about 50 times higher CACNA1C mRNA abundance than U251, but not in U251 cells, benidipine aggravated TTFields-triggered cell cycle arrest, intrinsic apoptosis and DNA degradation and showed a tendency to act additively to TTFields on induction of aneuploidy and attenuation of clonogenic survival. In U251, benidipine alone triggered intrinsic apoptosis and decreased clonogenic survival. The latter was reversed by TTFields.Finally, we defined whether or not TTFields-induced cell death may lower clonogenic survival which is the most relevant endpoint in oncology with regard to tumor relapse after therapy. Pre-plating colony formation assay  did not match the reported  optimal The 6-years follow up of the multicentric randomized prospective clinical trial analyzing temozolomide vs. temozolomide plus TTFields maintenance therapy suggests that in particular glioblastoma patients with methylated MGMT (O-6-methylguanine-DNA methyltransferase) promotor in the tumor benefit from TTFields therapy . This mi2+ channels that comprise L-type CACNA1C (Cav1.2) channels as further TTFields targets. The dihydropyridine calcium channel blocker benidipine acts on L-, T-, and N-type channels. Like in the present study, alternating current electromagnetic fields (ac-EMF) such as microwave or Wi-Fi have been demonstrated in several independent studies to activate Ca2+ entry pathway in various cell types. Notably, these pathways were in part sensitive to inhibitors of voltage-gated Ca2+ channels suggesting that the ac-EMF sensitivity of voltage-gated Ca2+ channels is a general phenomenon  and determine the resting free[Ca2+]i in these cells [2+ and PKC activity [2+ entry in TTFields-treated glioblastoma cells.The reported interference with DNA repair by TTFields  also sugview see ). Particse cells . Along tactivity . This mi2+-dependent signaling pathways [Taking into account that cell cycle , cell mipathways  an inter2+ channel blocker benidipine did not revert the observed cellular TTFields effects indicating that these effects most probably were not induced by TTFields-triggered benidipine-sensitive Ca2+ influx. Rather, benidipine aggravated (or showed the tendency to increase) the TTFields effects in T98G cells. Unexpectedly, such a concerted action of benidipine and TTFields was not observed in U251 cells although both glioblastoma lines exhibited comparable TTFields-induced Ca2+ entries that were both completely blocked by benidipine  and lowered clonogenic survival (both lines) indicating its anti-neoplastic action. Among the known benidipine targets, mRNA encoding the L-type Ca2+ channel CACNA1C (Cav1.2) was most abundant in both glioblastoma lines strongly suggesting that the observed benidipine effects were mediated at least in part via inhibition of CACNA1C. The functional significance of CACNA1C for the Ca2+ signaling of both glioblastoma cell lines can be deduced from the decline in resting Ca2+ concentration  or ∆Ψ2+ antagonists even in tumors with unknown TTFields responsiveness. Benidipine, which is in clinical use in Japan and India  provides a rationale to combine alternating electric field therapy with Cao, Japan ) as antio, Japan . The usufedipine  seems to2+- and electrosignaling, cell cycle progression, programmed cell death, and clonogenic survival of glioblastoma cells. Concerning the most relevant parameter in oncology that is the clonogenic survival, TTFields responsiveness differed markedly between the two human glioblastoma lines tested suggesting that also in the clinical situation the benefit of TTFields therapy may vary considerably between individual glioblastoma patients. Knowledge of the underlying molecular mechanisms might be used for therapy stratification in the future or for pharmacological intervention and improvement of the tumoricidal TTFields effects. One such potential approach evolves from the present study that disclosed that Cav channel activity contributes to cellular stress response to TTFields and Cav inhibition may augment the TTFields effects.TTFields act on several molecular targets/pathways thereby influencing Ca


RUNX1 encodes a Runt-related transcription factor that is critical for hematopoiesis. In this study, through a combinatorial molecular approach, we characterized a novel t translocation involving RUNX1 that was acquired during the progression of myelodysplastic syndrome to acute myeloid leukemia (AML) in a pediatric patient. We found that this translocation did not generate RUNX1 fusion but aberrantly upregulated RUNX1. This upregulation was attributed to the disruption of long-range chromatin interactions between the RUNX1 P2 promoter and a silencer in the first intron of the gene. Characterization of the silencer revealed a role of SNAG repressors and their corepressor LSD1/KDM1A in mediating the effect. Our findings suggest that chromosomal rearrangements may activate RUNX1 by perturbing its transcriptional control to contribute to AML pathogenesis, in keeping with an emerging oncogenic role of RUNX1 in leukemia.The online version of this article (10.1186/s12943-018-0881-2) contains supplementary material, which is available to authorized users. RUNX1 gene contains two functionally distinct promoters, the distal P1 and proximal P2, which are separated by a large first intron of ~ 160 kilobases (kb). RUNX1 is commonly disrupted by chromosomal translocations in hematological malignancies but the molecular consequences have only been characterized in less than half of the cases. It is generally believed that RUNX1 translocations generate oncogenic fusion proteins or truncate RUNX1 that interfere with wild-type RUNX1. Here, we reveal a novel mechanistic impact of a RUNX1 translocation in an acute myeloid leukemia (AML) case transformed from myelodysplastic syndrome (MDS).RUNX1 is a master regulator of hematopoiesis and its activity is tightly controlled at the transcriptional and post-transcriptional levels. The RUNX1 . Whole genome sequencing (WGS) on the relapsed AML revealed that chromosome 21 broke at intron 1 of RUNX1, whereas chromosome 5 broke at an intergenic region that is ~ 90 kb centromeric of SERF1A  and its disruption may perturb RUNX1 expression [RUNX1b/RUNX1a) in the relapsed AML BM, as compared to the t-negative MDS and post-transplant samples from the patient /RUNX1-RUNX1T1 with RUNX1 breakage at intron 5 and 7 with other cytogenetic aberrations or a normal karyotype at diagnosis) revealed only modest RUNX1  upregulation in the leukemia counterparts (mean 2.47-fold vs. 8.53-fold in our case)  examination showed focally prominent blastic infiltrations 15–20%) with dysplastic changes involving the erythroid and megakaryocytic lineages, suggestive of the diagnosis of advanced MDS. No cytogenetic abnormalities were detected. The patient was treated with intensive chemotherapy with initial response. However, blasts emerged 7 months later with the acquisition of a novel translocation tq13;q22), indicating disease transformation to AML . Collectively, these findings indicate that the RUNX1 P2 promoter is restrained by an intronic silencer involving SNAG repressors and their corepressor LSD1 in myeloid cells.Analyses of the ENCODE ChIA-PET  RNA polymerase II data from K562 cells revealed four regions in intron 1 that showed robust interactions with the P2 promoter /ETV6-RUNX1 found in B-lineage acute lymphoblastic leukemia. It was suggested that disruption of this intron might deregulate the RUNX1 promoters and cause RUNX1 overexpression in ETV6-RUNX1-positive patients without concurrent RUNX1 gain [RUNX1 transcription was suggested in a cryptic t involving breakage of the same intron in a familial platelet disorder (FPD)/AML case [RUNX1 breakpoint in our case lies between the silencer and P2, the t will disrupt this long-range control, resulting in unrestrained P2 transcription. It has been demonstrated that RUNX1 binds to two RUNX motifs in the P1 promoter to activate its transcription in hematopoietic cells [RUNX1c increase in our case ;21RUNX1 p1;q22/ETV6ic cells . It is tp1;q22/ETV6WT1 (Wilms tumor 1) mutations are rare in MDS but associated with AML transformation [WT1 mutations in the two AML but not the MDS samples  might cooperatively contribute to the AML transformation. Concordantly, we found that the overall RUNX1 expression is significantly increased in AML than MDS patients  on neighboring 21q22 and 5q13 genes could not be assessed.It remains to be determined if additional CREs and/or factors are involved in the LSD1-mediated RUNX1 deregulation by revealing that chromosomal translocations can also aberrantly upregulate RUNX1. This highlights the multifaceted impacts of chromosomal aberrations on RUNX1 and may open a new avenue for investigating RUNX1 alterations. Also, our findings support the emerging oncogenic role of RUNX1 in AML, suggesting that blocking RUNX1 activity may be a potential therapeutic approach. Lastly, the characterization of the RUNX1 silencer further underscores the complex transcriptional control of the gene and provides new insights into CRE functions.In conclusion, our findings uncover a novel mechanism of Additional file 1:Methods and Materials. including primers used in this study (Table S1) and supplementary references. (DOCX 32 kb)Additional file 2:Figure S1.RUNX1 expression in 14 pairs of leukemia/remission BM samples from pediatric AML patients. (DOCX 304 kb)Additional file 3:Table S2. Regions of intron 1 that interact with the RUNX1 P2 promoter as revealed from the K562 RNA polymerase II ChIA-PET data. (DOCX 26 kb)Additional file 4:Figure S2. Multiple alignments (Multiz) of the putative GFI1/GFI1B and SNAI1 binding sites in the silencer element. (DOCX 307 kb)Additional file 5:Figure S3. Knockdown of GFI1/GFI1B and SNAI1 alleviated the repressive effect of the silencer element. (DOCX 291 kb)Additional file 6:Figure S4. Dose-dependent upregulation of RUNX1 expression by the LSD1 inhibitor tranylcypromine (TCP). (DOCX 35 kb)Additional file 7:Figure S5. Orientation- and promoter-dependent properties of the RUNX1 intronic silencer. (DOCX 507 kb)Additional file 8:Figure S6.WT1 mutations in the AML but not MDS samples of the patient. (DOCX 1483 kb)Additional file 9:Figure S7.RUNX1 mRNA levels in MDS and AML patients. (DOCX 61 kb)


GXG2 was isolated and characterized by function-based screening strategy. Results of multiple alignments showed that the derived protein BglG shared 45.7% identities with reviewed β-glucosidases in the UniProtKB/Swiss-Prot database. Functional characterization of the β-glucosidase BglG indicated that BglG was a 468 aa protein with a molecular weight of 53.2 kDa. The BglG showed the highest activity in pH 7.0 at 35 °C and exhibited consistently high levels of activity within low temperatures ranging from 5 to 35 °C. The BglG appeared to be a psychrophilic enzyme. The values of Km, Vmax, kcat, and kcat/Km of recombinant BglG toward ρNPG were 1.1 mM, 1.4 µg/mL/min, 12.7 s−1, and 11.5 mM/s, respectively. The specific enzyme activity of BglG was 12.14 U/mg. The metal ion of Ca2+ and Fe3+ could stimulate the activity of BglG, whereas Mn2+ inhibited the activity. The cold-adapted β-glucosidase BglG displayed remarkable biochemical properties, making it a potential candidate for future industrial applications.The products of bacterial β-glucosidases with favorable cold-adapted properties have industrial applications. A psychrophilic β-glucosidase gene named  Fast Digest PstI and Fast Digest EcoRI, DNA ligase, Pfu DNA polymerase, and λDNA/HindIII DNA Ladder were all purchased from New England BioLabs . Esculin hydrate, ρNPG, and all chemical reagents were bought from Sigma-Aldrich, Inc. . Nickel–nitrilotriacetic acid (Ni–NTA) was bought from QIAGEN . Biospin Gel Extraction Kit, Biospin Bacteria Genomic DNA Extraction Kit, and Biospin PCR Purification Kit were all purchased from BioFlux .pGEM-3Zf (+), pETBlue-2, Exiguobacterium sp. GXG2 was isolated from subtropical soil samples in Nanning, Guangxi, China  and deposited at the China Center for Type Culture Collection with the accession number of CGMCC16605  and chemically transformed into E. coli DH5α. The fragments were spread on Luria–Bertani (LB) agar plate supplemented with 0.05% of ferric ammonium citrate (w/v), 100 μg/mL ampicillin and 0.2% esculin (w/v), IPTG, and X-gal at 37 °C. The colonies with black phenotype sequenced at Sangon Biotech Co., Ltd.  were considered to show β-glucosidase activity.In a previous study, a strain http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) was used to predict gene Open Reading Frames (ORFs). The similarity search for amino acids from ORFs was performed by the Basic Local Alignment Search Tool in the UniProtKB/Swiss-Pro database (https://www.uniprot.org). The websites ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) and CLUSTALW  were used for multiple sequence alignment. MEGA 6.0 software was used to construct a phylogenetic tree. The SWISS-MODEL server (https://www.swissmodel.expasy.org) and MODELLER program (version 9.18) were utilized for homology modeling. Four templates  were used to build the 3D structure of BglG. NAMD  pLacI were used as expression vector and strain, respectively, and cultured in LB medium containing ampicillin (100 μg/mL). Primers (B0F) CCGGAATTCTATGAAAATGCCAAAAGATTT and (B0R) TAACTGCAGTAGTTCAGCAGCACGTGTCG were used to verify the recombinant strain. The optimal temperature, IPTG concentration, and induction time were 20 °C, 0.8 mM, and 6 h, respectively. The induced OD of cells reached 0.6 at 600 nm, and the cells were collected by centrifugation at 9000 rpm for 20 min. The His60 Ni Gravity Column Purification Kit  was used the His-tagged protein purification with some modifications. Pre-cooled 2 mL His60 Ni ×Tractor Buffer was added to 100 mg cell pellet for resuspension and cell breakage by ultrasonication at 4 °C . The clear supernatant was collected by centrifuged at 12,000 rpm at 4 °C for 30 min. Add the sample to the equilibrated the His60 Ni Gravity Column (1 mL) and allow His-tagged protein to bind for 1 h at 4 °C. 10 column volumes of Elution Buffer was used for eluting and 500 μL fractions was collected at 4 °C according to the user manual protocols of the Kit. The targeted protein was analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The 6× His-tagged protein were detected by incubation with (1:2000) anti-6× His tag antibody (ab1187) . The electrophoresis instrument and film transfer instrument were purchased from Bio-Rad .2HPO4–citric acid buffer; pH 8.6–10.6, glycine–NaOH buffer; and pH 10.9–11.0, Na2HPO4–NaOH buffer) at 37 °C for 20 min. For pH stability, BglG was placed in different pH levels (pH 4.0–11.0) at 4 °C for 12 h, and enzyme activity was measured after the substrate ρNPG was added at 35 °C for 20 min. The effect of temperature on BglG activity was performed in a disodium hydrogen phosphate (Na2HPO4)–citrate buffer of pH 7.0 at 5–65 °C  for 20 min. For thermostability, BglG was placed in pH 7.0 buffer at 5–65 °C for 1 h, and β-glucosidase activity was measured after the substrate ρNPG was added at 35 °C for 20 min.The effect of pH on BglG activity was performed at 0.1 M buffer  and different concentrations of ethylenediaminetetraacetic acid  were added to the reaction system at 35 °C and pH 7.0 for 20 min. No metal ions and EDTA were added as a control.Equal volumes of different chloride metal ions . One unit (U) of β-glucosidase activity was defined as the amount of enzyme that releases 1 μmol ρNP per minute.The enzymatic reaction solution comprised pre-warmed 10 μL of 20 mM ρNP was determined. Moreover, the enzyme activity on laminaribiose, cellobiose, barley  β-d-glucan and soluble starch were determined by measuring the amount of reduced sugars with the dinitrosalicylic acid method.Specificity of the BglG was measured by incubating the purified protein in glycine–NaOH (pH 7.0) that contains 1 mM of aryl-glycosides and saccharides  at 35 °C for 20 min. Under the standard assay condition, the released The accession number of the BglG nucleotide sequence was AKA66375.1 in the GenBank database.Exiguobacterium sp. GXG2 containing 10,080 positive clones was constructed with restriction enzyme sites PstI and EcoRI. The entire library capacity was approximately 30 Mb, and the inserted DNA size was 3–10 kb. A substrate activity screening strategy was employed to obtain a positive clone. This clone, namely pGEMB0, was isolated from the metagenome library with black circle after incubated at 37 °C for 24 h  and similarity (59%) in the PDB database   , and B. polymyxa  ily Fig. a  pLacI as an expression host. Crude cellulolytic lysate containing recombinant BglG was purified by the Ni–NTA column after being induced by 0.8 mM IPTG for 6 h at 20 °C and subjected to SDS-PAGE    at pH 7.0 and 35 °C. Lineweaver–Burk plots were used to determine reaction kinetic parameters of purified BglG -β-d-glucan and souble starch.The relative hydrolysis rates of some artificial and natural substrates by purified BglG were determinate Table . BglG caBacillus subtilis (GenBank: CAB12135.1)  (GenBank: CAA42814.1)     with β-glucosidase BglC from Exiguobacterium sp.  were highly conserved in the active center of the GH1 family BglG through NAMD and VMD software               of BglG was 4.3-fold higher than that of β-glucosidase from soil metagenome  linkages substrates. It could not break β-glucans containing β, β and β linkages. It also do not hydrolyzed βGal, βXyl and αGlc glycosidic linkages. The specific enzyme activity of BglG was 5- and 303-folds higher than those of β-glucosidases from almonds and In this study, a β-glucosidase gene was firstly isolated and characterized by function-based screening strategy from subtropical soil microorganisms. The recombinant BglG was demonstrated as a member of the GH1 family and Glu163 and Glu373 are two key catalytic sites in the active center of the BglG according to the analysis results of physicochemical properties, key site prediction, homology modeling, and molecular docking. The detailed biochemical characterization of BglG had also been performed, and BglG had been found to show unexpectedly high activity across a low-temperature range (5–35 °C). This raw material also offered a possibility to improve the properties of BglG through protein engineering for further industrial demand.Additional file 1. Table S1. The results of homology modeling of BglG by multiple-template. Table S2. The evaluation of and multi-template model of BglG. Fig. S1. Functional screening of gene library. Fig. S2. Agarose gel electrophoresis of bglG gene. Fig. S3. The ramachandran plot of BglG multi-template model. Fig. S4. The PROSA evaluation of BglG multi-template model. Fig. S4. The PROSA evaluation of BglG multi-template model. Fig. S5. Compare the changes in surrounding residues of active sites E163 and E373 after mutant to glycine.