To measure the rate of patients receiving high dosage antipsychotics.To review the adherence to maximum recommended doses of antipsychotics as per the product information approved by Australian Therapeutic Goods Administration, product information approved by Medsafe  and Therapeutic Guidelines High dose antipsychotics or combination of antipsychotics are associated with significant adverse effects including QTc prolongation, arrhythmias, sudden cardiac death, seizures, increased incidence and severity of adverse effects, longer hospital stay and possibly increased mortality. High dose antipsychotic prescribing may arise as a result of EITHER single antipsychotic drug prescribed at a daily dose above the recommended limit (High Dose single drug) OR More than one antipsychotic prescribed concurrently where the sum of doses given expressed as a percentage of the SPC maximum of each drug exceeds 100% (High-Dose through the prescribing of multiple drugs).The data were gathered from all the drug charts for all patients admitted to HDU and Acute ward on 9th April 2019. The Audit standards were 1) Individual antipsychotic dose should be within recommended limit as 100% and 2) Combined antipsychotics should be within recommended limit as 100%Total number of patients on both the HDU and Acute wards = 33Total number of patients on antipsychotics = 30Number of patients on > 100% of recommended cumulative dosage = 13/30 = 43.3%Number of patients on > 100% maximum limits of regular antipsychotics = 3 = 10%Number of patients on > 100% maximum limits of PRN antipsychotics = 0/30Number of patients on 2 antipsychotic = 18/30 = 60%Number of patients on 3 antipsychotic = 8/30 = 26.6%Number of patients on 4 antipsychotic = 2/30 = 6.6%Out of the 30 patients on antipsychotics, almost half were on more than 100% of the recommended cumulative maximum limits of antipsychotics doses, almost 2/3rds were on 2 or more antipsychotic and a quarter on 3 or more. This can be associated with significant adverse effects including QTc prolongation, arrhythmias, sudden cardiac death, seizures, increased incidence and severity of adverse effects, longer hospital stay and possibly increased mortality. There is a need to review PRN antipsychotics prescribed as a norm, clear documentation and need for a protocol for increased vital sign monitoring for patients on high dose antipsychotic treatment.


Mutations in BRCA1 and BRCA2 genes are well-established risk factors of breast and ovarian cancer. In our former study, we observed that approximately 6% of unselected ovarian cancer patients in the region of Podkarpacie (South-East Poland) carry BRCA1 causative founder variants, which is significantly lower than in other regions of Poland. Therefore, it is deeply justified to do research based on the sequencing of whole BRCA1 and BRCA2 genes.BRCA1 and BRCA2 genes Next-Generation Sequencing study in all cases.We examined 158 consecutive unselected cases of ovarian cancer patients from the region of Podkarpacie. We performed BRCA1 or BRCA2 pathogenic mutations were found. BRCA1 pathogenic variants were detected in 11 of the 158 (7.0%) ovarian cancer cases. 10 of 11 (91%) detected BRCA1 mutations were founder mutations, detectable with the standard test used in Poland. BRCA2 pathogenic variants were found in 7 of the 158 (4.4%) cases. No BRCA2 pathogenic variants were founder mutations. The median age of patients at the diagnosis of the 18 hereditary ovarian cancers was 57.5 years.Altogether, in 18 of 158 (11.4%) ovarian cancer patients with BRCA1 or BRCA2 gene mutation carriers among patients with ovarian cancer from the Podkarpacie region is comparable to other regions of Poland. However, a significantly higher percentage of BRCA2 gene mutations was observed, that were not detectable with a standard test for detection of founder mutations. Diagnostics based only on testing the BRCA1/2 Polish founder mutations is characterized by relatively low sensitivity in the case of ovarian cancer patients from South-East Poland and should be supplemented by NGS study, in particular of the BRCA2 gene.The frequency of  BRCA1  gene [BRCA1 and BRCA2 mutation frequency and spectrum have been reported [BRCA1 or BRCA2 founder mutation carriers, and a slightly different spectrum of these mutations than in other regions of Poland [The incidence of ovarian cancer is approximately 3,600 cases annually in Poland . It has f Poland .BRCA1 and BRCA2 mutations in unselected ovarian cancer patients from the Region of Podkarpacie with the use of NGS, and establish an optimal algorithm for genetic testing of women diagnosed with ovarian cancer from this region.The aim of this study was to define the prevalence and spectrum of Ovarian cancer cases were identified from patients treated at a clinical base in the Department of Obstetrics and Gynecology of Fryderyk Chopin University Hospital No 1 in Rzeszow, Poland between January 2013 and January 2017. All patients were inhabitants of the South-East region of Poland. The study group consisted of 158 consecutive, newly diagnosed cases of ovarian cancer after surgical treatment, unselected for age or family history. The mean age of diagnosis was 58.5 years (range 22–84 years). The reference pathologist reviewed a representative slide from each cancer to confirm the diagnosis. 20% of patients were diagnosed in the I and II clinical stages according to FIGO, and 80% in III and IV. 14.8% of ovarian cancers showed pathological grading G1, 11.1 - G2, and 74.1 – G3. A cancer family history was obtained during an appointment with a clinical geneticist.BRCA1 and BRCA2 genes Next-Generation Sequencing (NGS) study in all cases.DNA was isolated from 5 to 10 ml of blood. We performed BRCA1 and BRCA2 genes were screened using the SureMASTR BRCA Screen Kit from Agilent Technologies. The SureMASTR BRCA Screen (Agilent Technologies) analyses the full coding regions of these genes. In brief, 50ng (2.5ng/µl) of genomic DNA  was used to amplify the target genes in a single-tube multiplex reaction. The obtained amplicon libraries were purified and diluted before single-tube universal PCR reaction to tag all amplicons with specific p5 and p7 adaptors. Each tagged amplicon library was purified to remove small residual DNA fragments, and the DNA library concentration was quantified using a Pre-proof High Sensitivity Qubit quantification kit (Life Technologies). Equimolar quantities of the individually tagged amplicon libraries were pooled, and the final sequencing library was normalized to a concentration of 4nM. Sequencing was performed on a MiniSeq platform (Illumina) using the MiniSeq Mid Output Kit, 2 × 150 cycles, to obtain reads for both strands. All detected pathogenic mutations and variants of unknown significance (VUS) were validated using Sanger sequencing. The conventional Sanger sequencing was performed with the use of BigDye terminator sequencing kit v3.1 (Life Technologies) on the ABI Prism 3130 genetic analyzer (Life Technologies) according to the manufacturer’s protocol.Bioinformatics analysis was performed using the software MASTR Reporter (Agilent Technologies). This analysis included read alignment to the human reference genome (Genome Reference Consortium GRCh37), variant calling, and visualization of the sequence reads. Variants above 40X coverage depth and with a minimum variant allele frequency of 5% for germline analysis are displayed in the software. Further filtering was applied to select germline variants which had a minimum of 100X coverage depth. Each sample passed quality control analysis.The clinical significance and the implications of variants were identified based on the annotations in public archives: ClinVar, Breast Cancer Information Core, Leiden Open Variation Database Online Mendelian Inheritance in Man (OMIM), Human Genome Variation Society (HGVS), and VarSome. Germline variants were classified according to the ACMG Standards and Guidelines for the Interpretation of Sequence Variants .BRCA1 or BRCA2 causative variant was found in 18 of 158 (11.4%) unselected ovarian cancer cases. A BRCA1 mutation was detected in 11 (7.0%) patients. The c.5266dupC mutation was the most common, it was diagnosed in six patients, followed by the c.181T > G mutation observed in three patients, and the c.676delT mutation in one patient. All carriers of these 3 mutations were diagnosed previously with the test based on detection of founder pathogenic variants characteristic for the Polish population [BRCA1 c.5346G > A mutation carrier (Table BRCA2 gene mutation was diagnosed in 7 (4.4%) unselected ovarian cancer cases. None of these BRCA2 mutations was a recurrent mutation characteristic for the Polish population. We also found in 3 patients, variants of unknown significance (VUS), all in the BRCA2 gene . The median age of diagnosis of the 18 hereditary ovarian cancers was 57.50 years (range 41–82 years), compared with a median age of diagnosis of 58.77 years (range 22–84 years) for the 140 cases without a mutation. However, the median age of diagnosis in BRCA1 carriers was lower than BRCA2 carriers − 55.8 vs. 60.1 years, respectively. A BRCA1 mutation was found in 3, and BRCA2 in 1 of 31 (together − 12.9%) women diagnosed with ovarian cancer at or under the age of 50 compared to 8 BRCA1 and 6 BRCA2 carriers of 127 (together − 11.0%) women diagnosed at a later age. Among the 18 women with ovarian cancer and a BRCA1 or BRCA2 mutation, ten reported a first- or second-degree relative with breast or ovarian cancer (55.5%), and there was only a slight difference between BRCA1 and BRCA2 carriers . A mutation was present in 25.6% (10/39) of ovarian cancer patients with a positive family history and in 6.7% (8/119) of women with a negative family history. A significant family history, defined as a presence of first- or second-degree relative with breast or ovarian cancer, was observed in 24.7% (39/158) of patients with ovarian cancer (Table A pulation . In addiBRCA1 or BRCA2 genes [BRCA1 causative founder variants was observed in about 10-13.5% of ovarian cancer patients [BRCA1/2 genes in the same group of 158 women affected with ovarian cancer and diagnosed 18 (11.4%) BRCA1/2 mutation carriers. The frequency of individual BRCA1/2 mutations observed in ovarian cancer patients is shown in Table BRCA1/2 founder mutations characteristic for the Polish population [BRCA1 and 7 carriers of the BRCA2 gene. These 8 mutations were detectable by whole sequencing only. Like in other regions of Poland, the most frequent mutation was the BRCA1 c.5266dupC mutation observed in 30% (6/18) of all carriers and the BRCA1 c.181T > G mutation found in 15% (3/18). Other 9 BRCA1/2 mutations were observed in single patients and are rare in the Polish population. In contrary to our former observation the frequency of BRCA1/2 mutation carriers in the group of ovarian cancer patients is only slightly lower than in other regions of Poland. However, we observed a significantly lower frequency of founder mutations, in particular, BRCA1 c.5266dupC and to a lesser extent BRCA1 c.181T > G. This phenomenon can be caused in general by the lower frequency of these mutations in South-East Poland. However, it should be noted that in this region the extensive genetic testing of BRCA1/2 genes has been carried out in patients with ovarian and breast cancer, as well as, in healthy patients since the year 2000. The testing focused mainly on the detection of founder mutations. As a result, several hundred families with BRCA1 founder mutations have been diagnosed so far and several hundred prophylactic adnexectomies have been performed. It should be taken into account that these BRCA1 founder mutations carriers were thus protected against ovarian cancer, and therefore, we observe their lower representation among BRCA1/2 mutation carriers who have now developed ovarian cancer.The region of Podkarpacie is located in the South-East corner of Poland, bordering Ukraine and Slovakia. In our previous study, we identified 10 of 158 (6.3%) of unselected cases of ovarian cancer from this region carried one of 13 founder mutations in the A2 genes . This ispulation . In addiBRCA1 mutation was 55.8 years, and of the 7 patients with BRCA2 mutation was 60.1 years. In both groups, the mean age at diagnosis was slightly higher than the observed in BRCA1/2 carriers from other regions of Poland [BRCA1/2 carriers. However, for non-carriers, the mean age at diagnosis was similar in the region of Podkarpacie and the rest of Poland (58.77 vs. 56.2–62.3 years) [The mean age at diagnosis in the 11 cases with BRCA1 as well as the BRCA2 gene. It can be explained by the relatively larger number of family members in an average family, in this region. However, the frequency of BRCA1/2 mutation carriers with negative family history is so high (44.5%), both in groups with recurrent founder mutations as well as with non-founder mutations, that we cannot recommend limiting performing the BRCA1 and BRCA2 gene testing based on NGS to cases with a burdened family history only. Also, the ovarian cancer age of onset is not a factor facilitating the qualification for this study. Taking into account our observations, it should be stated that performing a test based on the detection of Polish founder mutations in ovarian cancer patients from the Podkarpacie region is associated with relatively low sensitivity (55.5%). In turn, performing the NGS test in all subsequent patients with ovarian cancer is associated with a significant increase in costs. Application of NGS tests only in familial cases is associated also with low sensitivity of 55.5%. One of the compromise solutions would be to perform a standard genetic test based on the detection of founder mutations in all patients, and then if no mutation is detected, perform NGS in cases with a family history. With this algorithm of procedure, the sensitivity of detecting the BRCA1/2 gene mutation in ovarian cancer patients would increase to 77.8%, at a relatively low cost.We observed strong family history in 10 of 18 (55.5%) mutation carriers, which is slightly more frequent than in other regions –8. This BRCA1/2 gene is routinely tested using the NGS method in DNA extracted from tumour cells. It seems, therefore, that the most justifiable algorithm for detecting a germinal mutation in these patients is to start testing BRCA1/2 genes using the NGS method in DNA extracted from tumour cells. Then, if a mutation is found, the test should be performed in the patient’s peripheral blood to verify whether it is a germline or somatic mutation. If germline mutation is confirmed, the study should be extended to other relatives. However, if such a protocol is used, it should be taken into account that up to nearly 10% of cancer patients with negative NGS results performed in DNA isolated from neoplastic tissue cells, in fact, may carry germline mutation [However, taking into account the current diagnostic standards of patients with ovarian cancer in the context of determining the optimal treatment and qualifying patients for treatment with PARP inhibitors, the mutation . Lincolnmutation  indicatemutation , 13; (iimutation –17, (iiimutation . TherefoBRCA1 or BRCA2 causative variants. We found a significantly higher percentage of BRCA2 gene mutations, which are not detectable with a standard test for founder mutations. Diagnostics based only on testing the BRCA1/2 Polish founder mutations is characterized by relatively low sensitivity in the case of ovarian cancer patients from South-East Poland and should be supplemented by NGS study, in particular of the BRCA2 gene.Approximately 11% of unselected ovarian cancer patients in the region of Podkarpacie carry a


Scientific Reports 10.1038/s41598-022-07099-2, published online 24 February 2022Correction to: This Article contained a typographical error in the Results section.“The direct cause of both accidents Please check and confirm the edit and layis shown in Table 1, and the local workplace factors and organizational factors shown in Tables 2 and 3 have been clarified according to each SHEL element classification.”now reads:“The direct cause of both accidents is shown in Table 1, and the local workplace factors and organizational factors shown in Tables 2 and 3 have been clarified according to each SHEL element classification.”The original Article has been corrected.


The data in Table 2 regarding population aged younger than 20 years, population aged 70 years or older, and population density should have been calculated using the same measurement units as in Table 1. Table 1 has also been amended to fix incorrect values that were given for population density. This article has been corrected.In the Original Investigation titled “Association of Social and Economic Inequality With Coronavirus Disease 2019 Incidence and Mortality Across US Counties,”


In an articleIn Figure 1(A), the strips from HepG2 and Huh7 cell lines have been replaced by new strips. The correct strips in Figure 1(A) are shown below.In Figure 5(C), the colony image of Sorafenib + DZNep treated Huh7 cells in colony formation assay was accidently misplaced and has been replaced by the correct image now. The correct colony image of Sorafenib + DZNep treated Huh7 cells is shown in the figure below.In Figure 5(D), the TUNEL staining and DAPI staining image of DMSO treated 7404 cells in TUNEL assay was accidently misplaced, and has been replaced by the correct image now. The correct the TUNEL staining and DAPI staining image of DMSO treated 7404 cells in TUNEL assay in the figure is shown below.


For the CH4(70%)–H2(30%)mixture, the flame in the porous medium can be modulated by fluctuationsbetween 0 and 30% of steady methane flow without any noticeable flamedestabilization.Fluctuations in thefuel flow rate may occur in practical combustionsystems and result in flame destabilization. This is particularlyproblematic in lean and ultralean modes of burner operation. In thisstudy, the response of a ceramic porous burner to fluctuations inthe flow rate of different blends of methane and hydrogen is investigatedexperimentally. Prior to injection into the porous burner, the fuelblend is premixed with air at equivalence ratios below 0.275. Thefuel streams are measured and controlled separately by programmablemass flow controllers, which impose sinusoidal fluctuations on theflow rates. To replicate realistic fluctuations in the fuel flow rate,the period of oscillations is chosen to be on the order of minutes.The temperature inside the ceramic foam is measured using five thermocoupleslocated at the center of the working section of the burner. The flameembedded in porous media is imaged while the fuel flow is modulated.Analysis of the flame pictures and temperature traces shows that theforced oscillation of the fuel mixture leads to flame movement withinthe burner. This movement is found to act in accordance with the fluctuationsin methane and hydrogen flows for both CH The mixturewas ignited upstream of a quartz tube containing a porous medium,which also provided partial optical access to the flame.39 The flame movement was recorded via a digitalcamera, and the temperature was measured by a thermocouple at thedesignated parameters of the hydrogen–air mixture. Kakutkinaet al.39 found that for a 70% hydrogenmixture, the flame propagates upstream at a distance of 100 mm in2000 s, and the maximum temperature recorded was around 950 K at approximately1750 s under steady-state conditions. Fuel interchangeability wasstudied by Alavandi and Agrawal40 by combustinglean blends of hydrogen-syngas and methane fuel mixtures inside aporous burner. The air flow rate was kept constant for all tests,while the methane concentration was lowered for each test as the hydrogenand carbon monoxide fuel rates were adjusted to produce the requiredthermal power under steady-state conditions. The authors40 reported reduced carbon monoxide and nitrogenoxide emissions at any flame temperature for hydrogen and carbon monoxidemixtures compared to those produced by a pure methane flame.Kakutkinaet al.41 studied pollutantemissions when hydrogen was added to a natural gas mixture insidea porous burner. The burner was first operated with a natural gas–airmixture; once the combustion was stabilized, a gradual addition ofhydrogen was made. The experiments were conducted for 0.3 ≤ϕ ≤ 0.95, 100 ≤ P ≤ 700and an interchangeable hydrogen concentration of up to 100% withinthe natural-gas and hydrogen fuel composition. Gauthier et al.41 reported that as natural gas is slowly replacedby hydrogen a reduction in carbon monoxide, carbon dioxide, and nitrogenoxides took place. Also, they found that when the hydrogen contentexceeds 80%, the flame becomes unstable.Gauthier et al.34 experimentally examinedthe combustion of a premixed hydrogen–air mixture by varyingthe size of the combustion chamber inside the porous burner. The purposeof the study was to monitor the flame stability with the inclusionand exclusion of porous media in the combustion chamber. The authors34 chose a stainless-steel mesh to represent theporous media, whereby the hydrogen–air mixture was operatedfor different mass flow rates and equivalence ratios. Peng et al.34 found that as the diameter of combustor chamberdiminished, the flame front within the porous media enlarged acrossthe flow direction. Also, with the insertion of porous media, an increasein heat transfer accelerated the combustion process. Inspired by Alavandiand Agrawal,40 Arrieta et al.42 studied the combustion of mixtures of methaneand syngas inside a porous burner. Their experiments were focusedon the emissions of CO and NOx, flame stability responseto assigned thermal power, and the effects of volume fraction of thesyngas mixtures under steady-state conditions. Arrieta et al.42 used methane as the basic fuel for all testswhere the hydrogen to carbon monoxide ratio was varied. It was concludedthat the addition of hydrogen-rich syngas to methane did not impactthe flame stability or temperature profile drastically. Yet, a considerablereduction in CO and NOx emissions was reported.Peng et al.38 Upon introduction of fluctuations, the flamefeatured hydrodynamic motion inside the porous foam. It was observedthat there were certain amplitude and frequency of flow modulationunder which the flame could survive.38 Asthe characteristics of CH4–H2 blendsare quite distinctive to those of CH4–CO2, the burner stability for combustion of methane and hydrogen blendsneeds to be re-examined. This is particularly due to the high reactivityof hydrogen which can significantly affect flame blow-off and extinction.Themost recent work of the current authors showed that an ultraleanmixture of methane and carbon dioxide burning inside a porous foamcould strongly respond to fluctuations in the methane flow rate.4–H2 mixtures subject to inlet flow disturbances under ultraleanconditions. In an attempt to address this shortcoming, the currentwork investigates experimentally the unsteady ultralean hydrogen combustionof different blends of methane and hydrogen.As heat transfer dominates combustion in porous media, the latteris expected to be influenced by variations in the inlet flow. Thisis particularly the case in ultralean combustion in which changesin the temperature can have a profound effect on the flame stability.Nevertheless, as seen in the preceding survey of literature, currently,there is no systematic experimental study on CH22.1A schematicrepresentation of the experimental setup is illustratedin 2.1.12O3 foam (20 ppi—ε ≈0.47) positioned nearest to the inlet valve preceded by a funnel-shapedSiC ceramic foam (20 ppi—ε ≈ 0.47). This was followedby a stack of low-density SiC foams (10 ppi—ε ≈0.72) completing the preheating region. The combustion region (visiblethrough the quartz glass tube) consisted of predominately high-densitySiC foams (20 ppi—ε ≈ 0.47). Two layers of low-densitySiC foams (10 ppi—ε ≈ 0.72) were placed near thecenter of the combustion region to provide further flame stabilityas illustrated in The combustion compartment was made up of two separate sections:the combustion and preheating regions. Both regions consisted of porousceramic layers stacked one atop the other vertically. The preheatingsection (designed to reduce the probability of flashback) entailedan Al2.1.2An openingwas created between the internal side of the outer casing and theexternal side of the combustion compartment for water cooling to takeplace. The vacant gap acted as a water reservoir with an integratedinlet and outlet within the porous burner. The water coolant systemdrew cool water from the water source filling the water reservoirfrom the inlet located near the bottom of the burner. Once the reservoirwas full, the water departed from the outlet (located near the topof the burner) warranting a thorough cooling of the stainless-steelburner.2.1.3The mixtureentering the burner consisted of a blend of hydrogen and methane dilutedwith air. Fuel was supplied from the corresponding gas cylinders anddigital mass flow controllers (MFCs) equipped with manually operatedvalves. Moisture was removed from the compressed air with the useof an air filter, and the air was supplied in an arrangement similarto that of the fuel where a quarter-turn hand valve was installedbefore the air MFCs. Flow Vision SC software was obtained from ALICATto vary the mass flow rate of each MFC using a computer. The programmableMFCs from ALICAT Scientific operated under an uncertainty of ±0.6%for both air and fuel. A number of MFCs were installed with relevantoperational ranges 1a for fu2.1.42 and ±20ppm for CO. A full high-definition video camera (1920 × 1080)was mounted ≈1.5 m from the burner to record the flame performanceand migration.2.243 was noted to be44 is given byThe equivalenceratio2, and NOx of the cases described in x emissions arealmost negligible. This is to be expected as the current low-temperaturecombustion system strongly suppresses formation of thermal NOx. Once the flame had been stabilized, the concluding temperaturevalues were noted from Pico software.After the flame was stabilized, as describedin Uncertainty errorwas also calculated to ensure adequate accuracyof the experimental procedure during flow modulation. This includedthe uncertainty error for MFCs and thermocouples. To determine this,thermocouple values were used from case 6x where T3 reported the highesttemperature (1384 K). Therefore, the T3 uncertainty error was assumedacross all five thermocouples. Combined with the supplied MFC manufacturererror (±0.6%), the total accumulative uncertainty error in flowmeasurements = ±2.7%.2.3x-axis to determine the flame migrationin the y-direction. Every picture was transformedfrom an RGB image to black and white where a brightness criterionwas introduced for flame detection. The luminosity value for eachconcentration of fuel varied thus authenticated by visual validation.As the brightness conditions were fulfilled, the vertical positionof the parameter was determined (i.e. where the flame had been identified)at the specified reference points. The designated values were reducedby filtering out only the top and bottom y-location.These values served to define the upper and lower section of the flameacross all reference points. This procedure reoccurred until all pictureshad been processed by the code. The final top and bottom positionsof the brightness criterion were then plotted with respect to time,illustrating migration of the flame vertically within the porous mediaalong the five reference points over a full oscillatory cycle.An in-depth imageanalysis was carried out to observe the change in flame position.The video recording of an entire oscillation of 60 s was cut out fromthe footage of each case described in 3The burner was operated undera steady state as well as fluctuatingfuel flow. Here, the outcomes of these two modes of operation arepresented separately.3.118 Flame stabilization is most commonly attainedwhen the flame speed matches the upstream flow velocity of air andfuel mixture.2738 have extensivelydiscussed the response of methane and biogas mixtures within the burnershown in 4(90%) + H2(10%) mixture exhibits a larger thermal power when comparedto its counterpart CH4(70%) + H2(30%); whichis predominantly due to its larger concentration of methane as (perunit volume) methane has a higher enthalpy of combustion comparedto hydrogen. Further, CH4(90%) + H2(10%) isable to operate at ϕ = 0.25 as hydrogen provides a higher flametemperature than methane alone to preheat the incoming cold reactantsand avoid flame extinction.In orderto achieve stable combustion inside the ceramic foam, a fine balancebetween heat production, heat deficit, and heat recirculation is required.4(70%)–H2(30%) mixture produces the highest temperature(1340 K), followed by CH4 (1291 K) and then biogas (1233K). This is due to the addition of hydrogen to methane, as hydrogenproduces a greater flame temperature when compared to pure methane.It is also noted that the peak temperature for each mixture is detectedat a different thermocouple point as the flame location within theburner differs between the fuel mixtures. In 4(70%)–H2(30%) mixture. The CH4(70%)–H2(30%) mixture shows 85% reduction in carbon monoxide emissions incontrast to CH4. This could be primarily attributed tothe higher flame temperatures of CH4–H2 blends compared to those of CH4 and biogas. High temperaturefacilitates the oxidation of carbon monoxide into carbon dioxide andthus reduces CO emissions.3.24(90%)–H2(10%) cases before superimposing oscillatory disturbancesat the inlet. 4(90%)–H2(10%) mixture subject to an oscillatory disturbance introducedon the methane flow with an amplitude of 10% of its steady value,over a period of 60 s. The thermocouples were used to record the temperatureof each case where the cut-off temperature to detect flashback wasdefined as T = 773.15 K. The temporal variation ofmethane flow leads to a distinct movement of the reaction region withinthe ceramic foam. This flame movement was video recorded –H2(10%) mixture. As a result, during the trough at an amplitudeof 50% of methane oscillation, the equivalence ratio plummets to 0.1413.Due to this drop, the porous burner struggles to retain heat evenafter supply of the excess methane to the combustion chamber. A similarphenomenon was also reported by Habib et al.38 while operating the same burner on biogas. Yet, it was observedthat an amplitude of 30% of the fuel flow rate for biogas was sufficientto incur greater heat loss. Further, for case 3x, an overall temperaturereduction is observed at thermocouples 3, 4, and 5 with time as thepreheating region receives minimal quantities of heat from the combustionregion. As this continuous cycle of heat loss progresses, the substantialreduction in temperature rules out the possibility of sustaining aflame within the pores of the ceramic foam. Yet, a thicker flame isvisible as a result of greater forcing amplitude despite very littleflame motion.46 The heat loss can be monitoredby the reduction in flame visibility in 4(90%)–H2(10%) mixture was subjectedto inlet sinusoidal disturbances. 4(90%)–H2(10%) mixture velocity even with the hydrogen amplitude set as highas 50%. Therefore, it is noted that with the exception of the secondthermocouple, most thermocouples are insensitive to the oscillationsin hydrogen flow with the third thermocouple recording the highesttemperature (1384 K) early on in the experiment. It can be seen thatthe porous burner withstands the introduced inlet disturbances withoutany visible signs of flame flashback or blow-off. A distinct contrastin the temperature trace of the second thermocouple emerges betweenthe flow modulation of methane and hydrogen. The temperature tracein 4(90%)–H2(10%) mixture shows insignificantflame motion during the eighth cycle. It is noted that in this case,the brightness and color of the flame are not as vivid as relatedto methane modulation due to hydrogen flame being nearly invisible.Next, the fuel concentration of hydrogen in theCH4(70%)–H2(30%) mixture when methane flowis modulated with an amplitude of 10%. The starting point before introducingdisturbances at the inlet was selected to be case 1b for additionalsafety. As the amplitude of oscillation is 10% for the methane flowrate, the equivalence ratio increases to 0.3 at the peak point ofthe oscillatory cycle, much greater than the value at which steadyCH4(70%)–H2(30%) mixture operates stably.However, the local flame is choked when the equivalence ratio fallsto 0.25 at the lowest point of the oscillatory cycle. Still, as theoscillatory cycle period is limited to 60 s, the system not only holdsonto the heat but as the methane flow rate augments, the excess enthalpycombustion is fast-tracked. Consequently, in 38 as thesame porous burner was operated solely on a CH4 mixture.However, despite the CH4 mixture having an identical amplitudeof oscillation, it was noted that the flashback occurred due to alonger oscillation cycle (180 s). 4(70%)–H2(30%) mixture; convection amid the fluid and solid occurs and warmsthe cold reactants. Yet, due to the existence of the low porosityceramic foam downstream of the combustion region, conduction is acceleratedin response to significant methane fluctuations. 4(70%)–H2(30%) flame as the amplitude of hydrogen flow is modulated by 10%. 4(70%)–H2(30%)mixture. In this case, the porous burner manages to withstand aroundeight oscillatory cycles (y cycles 11a befor4(90%)–H2(10%)and CH4(70%)–H2(30%) mixtures. This comparisonprovides further clarity to the results reported earlier. 4 modulation amplitude. Similarly, H2 fuel modulation outputs larger temperature in comparison to CH4 modulation without directly responding to the oscillatorypattern. Case 5x and case 6x display a negligible temperature change. 4(70%)–H2(30%) mixture as theflame has moved further upstream with the greater addition of H2. As a disturbance is superimposed on the fuel inlet, thetemperature trace appears to follow a monotonic increase with theincrease of H2 amplitude. However, this pattern is notobserved as the CH4 amplitude was increased. Case 1y andcase 3y show greater temperature readings and eventually result inflashback albeit case 2y which maintains stable operation with a lowertemperature.4(90%)–H2(10%) and CH4(70%)–H2(30%) mixtures.Once again, the flame thickness in this figure was inferred throughthe image processing technique detailed in 4(90%)–H2(10%) mixture, it can be seen that as the methane amplitudeis increased, mid-way, the flame thickness contracts 13% before postinga gradual increase of 4%. An inverse behavior is observed for hydrogenmodulation, as the amplitude increases so do the flame thickness mid-waybut then contracts at the maximum amplitude value. However, for theCH4(70%)–H2(30%) mixture a monotonicincrease in flame thickness is detected for an increase in amplitudefor both methane and hydrogen, approximately, 300% overall. The exactreason for this behavior is not immediately obvious, and it callsfor further experimental and numerical studies.44(90%)–H2(10%) and CH4(70%)–H2(30%) mixtures were incorporated as the fuel. The inlet sinusoidaldisturbances were imposed on the hydrogen and methane flows by programmableMFCs. The thermal response of the system was monitored by using thermocouplespositioned at the center of the burner in different axial locationsand by detecting the flame motion using image processing techniques.It was found that under steady-state conditions, CH4(90%)–H2(10%) could operate as low as ϕ = 0.25. Additionally,the CH4(90%)–H2(10%) mixture producedgreater carbon monoxide emissions when compared to the CH4(70%)–H2(30%) mixture. As both mixtures were subjectto unsteady inlet flow of hydrogen and methane, the burner remainedstable for the CH4(90%)–H2(10%) mixturewhen hydrogen was oscillated between 0 and 50% and for methane between0 and 10% over a period of 60 s. Similarly, the burner remained stablefor the CH4(70%)–H2(30%) mixture whenhydrogen flow was fluctuated at 0–30% of its steady flow rateand methane fluctuated at 30% for the identical oscillatory period.For both mixtures by in large, it was observed that the flame movementcorresponds to the dynamics of the induced disturbances at the inlet.The flame thickness within CH4(70%)–H2(30%) mixture was proven to be far greater when compared to the CH4(90%)–H2(10%) mixture as the hydrogen andmethane flows were amplified. Lastly, both fuel mixtures were notedto be rather insensitive to hydrogen fluctuation beneath 30% amplitude.A custom-designed porous burner was used in an experimental investigationof ultralean combustion to investigate the burner response to theoscillations superimposed on the fuel flow. The work was motivatedby the possibility of temporal changes in the chemical compositionof hydrogen containing fuels including blends of methane and hydrogen.Such variations can strongly influence flame stabilization and leadto flame extinction/flashback. It is therefore important to analyzethe burner response to imposed oscillations on the fuel streams. CH


S3 (currently Appendix S2) of the Supplementary Material erroneously skipped blank, and the subsequent rows 43–67 (pages 10–11) swapped during the revision process. The correct version (Appendix S2) of the Supplementary Material is provided in this correction.The third cell in Row 42 in Appendix The original article has been corrected.Supplementary file1 (PDF 1202 kb)Below is the link to the electronic supplementary material.


Cell Death Discovery 10.1038/s41420-018-0125-7, published online 21 November 2018Correction to: The original version of this article unfortunately contained mistakes in Figs. 1 and 2. Preliminary experiments with a new batch of neuro2a cells from atcc suggests that neuro2a cells behave similarly to the mn9d cells used in the manuscript (they are sensitive to Mcl1 inhibition with umi-77 and Mcl1 appears to have the same molecular weight as in mn9d cells). The corrected figures can be found below.


This article has been corrected: The authors recently found errors inFigure 2B and 5D - the images for the "KYSE-150" groupwere incorrect. The authors corrected panels 2B and 5D in Figure 2 andFigure 5 by using representative images from the original sets of experiments.These alterations do not affect the results or conclusions of this work. The authors wouldlike to apologize for any inconvenience caused.Figures 2 and 5 are presented below.New


Robo2 resulted in misguidance of dI1i axons, whereas dI1c axons remained unperturbed within the mantle zone and ventral commissure. Further, Robo2 profoundly influenced dI1 cell body migration, a feature that was partly dependent on Slit2 signaling. These data suggest that dI1 neurons are dependent on Robo2 for their organization. This work integrated with the field support of a model whereby canonical Robo2 vs. non-canonical Robo3 receptor expression facilitates projection neurons derived from a common precursor domain to read out the tissue environment uniquely giving rise to correct anatomical organization.Sensory information relayed to the brain is dependent on complex, yet precise spatial organization of neurons. This anatomical complexity is generated during development from a surprisingly small number of neural stem cell domains. This raises the question of how neurons derived from a common precursor domain respond uniquely to their environment to elaborate correct spatial organization and connectivity. We addressed this question by exploiting genetically labeled mouse embryonic dorsal interneuron 1 (dI1) neurons that are derived from a common precursor domain and give rise to spinal projection neurons with distinct organization of cell bodies with axons projecting either commissurally (dI1c) or ipsilaterally (dI1i). In this study, we examined how the guidance receptor, Robo2, which is a canonical Robo receptor, influenced dI1 guidance during embryonic development. Robo2 was enriched in embryonic dI1i neurons, and loss of  The physiological function of the nervous system is dependent on the precise spatial connectivity of a diverse range of neural populations. This mature spatial organization originates from a relatively small number of progenitor domains, raising the broad question of how neurons derived from a common precursor origin and environment elaborate the spatial organization required for their later functional connectivity.During development, patterned multipotent neural stem cells differentially express key transcription factors instructing a cascade of events leading to their differentiation. Developing neurons subsequently migrate and grow in response to cues in their environment in a subtype-specific manner. This is achieved by the differential expression and localization of a small number of ligand/receptor molecular pathways used in various combinatorial codes together with various adapter molecules and gating signaling molecules to elicit specific responses. These broadly evolutionarily conserved pathways include the Slit/Robo, Eph/Ephrin, Semaphorin/plexin, neuropilin and Netrin/DCC, Uncs signaling, in addition to growth factors . Of thesRobo2 gene in mice resulted in misguidance of dI1 cell bodies and axons within the embryonic mantle zone. We further found that the misprojecting axons in Robo2 mutant embryos were dI1i neurons whereas pre-crossing and crossing dI1c neurons appeared unperturbed. This Robo2 phenotype was marginally enhanced by the loss of Robo1 and was partially phenocopied by the loss of Slit2. This indicated that Robo1 and Robo2 were not redundant in this context and that this phenotypic effect was elaborated at least in part through canonical Slit/Robo signaling. Overall, these findings taken together with previous results showing that non-canonical Robo3 controls dI1c axon guidance reveal a mechanism whereby Robo2 and Robo3 are differentially enriched at a key anatomical divergence point and result in the ability of dI1i and dI1c neurons derived from a common precursor origin to respond uniquely to their environment to elaborate the neural organization needed for their latter function.Overall, good progress has been made in understanding how Robo signaling and other molecular pathways promote correct guidance and organization of individual classes of neurons. However, in mammals the molecular logic of how this is gated by neurons derived from a common precursor domain or more broadly from multipotent stem cells remains poorly understood. The phenotypic antagonism between canonical vs. non-canonical Robo signaling could serve as a potential mammalian evolved mechanism to drive anatomical diversity from a common precursor domain; however, this remains to be examined. In two of the major systems used to probe neural diversification, the cortex, and the retina, non-canonical Robo3 signaling does not appear to play a major role in commissural axon formation . TherefoWild type SvEv129, GFPBarhl2, LACZMath1, +/–:Robo2+/–Robo1, Robo2+/–, and Slit2+/– mice . The following primary antibodies were used: chicken α-GFP , chicken α-GFP , rabbit α-Robo1 antibody produced in this paper , rabbit α-Robo1 . Z-stack photomicrographs (20×) were acquired using the Leica SP8 Falcon confocal microscope. Maximum projection images were generated using ImageJ, and the region of interest (ROI) was traced to demarcate dl1i and dl1c populations based on anatomy. The ratio between dl1i/dl1c of Robo1, Robo2, or Robo3 pixel intensity was quantified. Statistical analysis was performed as follows: Using Prism software, a one-way ANOVA Kruskal–Wallis non-parametric test followed by Dunn’s multiple-comparison analysis was performed.E12.5 spinal cord tissue from + neuron migration phenotype, transverse brachial sections of E12.5 +/+:Barhl2GFPRobo2 , +/–:Barhl2GFPRobo2 , –/–:Barhl2GFPRobo2 , Slit+/+:Math1LacZ2 , and Slit–/–:Math1LacZ2 , +/+:Robo2+/+:Barhl2GFPRobo1 , –/+:Robo2–/+:Barhl2GFPRobo1 , and –/–:Robo2–/–:Barhl2GFPRobo1  embryos were immunohistochemically labeled with Barhl2 to label dI1 neurons. From the images collected, a grid of 8 times 14 bins was superimposed on the images (Photoshop) so that the grid fitted the dorso-ventral and medial-lateral extreme of each spinal cord hemi-section analyzed  and –/–:Barhl2GFPRobo2  embryos were immunohistochemically labeled with the Lhx9 antibody. From the images collected, the dorso-ventral position of the main cohort of Lhx9+ neurons was measured, relative to the dorso-ventral length of the spinal cord in the section being examined. This percentage value was then averaged for control embryos which establish an average position of the control Lhx9+ neuron. This was 38% for control embryos (n = 7). A 38% ventral cutoff line was positioned on all the spinal cord images of control and mutant embryos , +/–:Barhl2GFPRobo2 , and –/–:Barhl2GFPRobo2  embryos were immunohistochemically labeled with Robo3 to label commissural axons and GFP to label dI1 neurons and were imaged with a Leica SP8 Falcon confocal microscope. Quantification was performed on the maximal projection of Z-stack images. Within ImageJ, two fixed-size boxes were applied to each image, Box 2 within the ventral mantle zone and Box 1 within the ventral commissure , the pixel density was measured within a defined region where dI1 axons and cell bodies were normally absent or observed at a low frequency in control embryos. In short, the area to be analyzed was first defined to exclude cell bodies on all sections analyzed. First, a rectangular region was defined which was delimited by the midline on the medial side and by the bottom of the floor plate on the ventral side , +/–:Barhl2GFPRobo2 (n = 6), –/–:Barhl2GFPRobo2 , +/+:Robo2+/+:Barhl2GFPRobo1 , –/+:Robo2–/+:Barhl2GFPRobo1 , and –/–:Robo2–/–:Barhl2GFPRobo1  embryos. Values from a single embryo were averaged to generate a value for that embryo. The “n values” refer to the number of embryos analyzed for each genotypic group. Values for each embryo analyzed were averaged (mean) for all embryos of a single genotype. The statistical analysis was performed using GraphPad (Prism) software. The data distribution was non-parametric, and the following statistical test was performed to examine statistical differences: Kruskal–Wallis test followed by Dunn’s multiple-comparison test using Prism software.Photomicrographs of E11.5 were imaged. The region of interest was defined by delimiting the white mater tracts as the outer border and commissural axons tract as the inner boundry respectively. Z-stack photomicrographs (20×) of E12.5 mouse embryonic spinal cord tissue immunohistochemically labeled with GFP (dI1 neurons) were acquired at subthreshold pixel intensity with respect to GFPwild type or GFPBarhl2 mouse embryos, a previously characterized transgenic mouse line which expresses GFP in both dI1 neuron axons and cell bodies  vs. commissural (Robo2low/Robo3+) dI1 neurons whereas Robo1 was expressed in both dI1i and di1c neurons with a modestly higher expression dI1c compared with dI1i neurons , Robo2 heterozygote (+/–:Barhl2GFPRobo2), and control (GFPBarhl2) embryos.Given the enrichment of Robo2 in dI1i neurons, we next explored the possibility whether the canonical Robo2 receptor influenced dI1 guidance. For this analysis, we crossed the Robo2 mutant and control embryos with a transcription factor delineating dI1 neuron nuclei, Barhl2  and control (LacZMath1) embryos to determine whether a similar phenotype was observed between Slit2 and Robo2 mutant embryos. Equivalent to the analysis for the Robo2 mutant embryos, we analyzed the distribution of Barhl2 nuclei in Slit2 mutant embryos. We found a notable medial shift in the population distribution on Slit2 mutant embryos, consistent with a medial shift in the dI1i neural population  and measured pixel density in defined regions , supporting the notion that the phenotypes were derived from the ipsilateral population. Further, we observed that in the Robo2 mutant embryos, commissural axon projections were projecting relatively normally within the mantel zone at E11.5 and E12.5, suggesting that dIc neurons within the mantle zone remained unperturbed  and GFP+ (dI1 neurons) immunolabeled axons within the mantle zone and ventral commissure using pixel intensity as a proxy for axon density. The ratio of the pre-crossing/crossing , Robo1/2 double heterozygote (+/–:Robo2+/–:Barhl2GFPRobo1), and control (GFPBarhl2) embryos. Using comparable analysis to that of Robo2 mutant embryos, we observed both axon guidance and dI1 neuron migration misguidance phenotypes in Robo1/2 double mutant compared with control embryos  does not produce a mantle zone or ventral commissure commissural axon phenotype unless Robo1 is modulated at the same time  within the mantel zone are gated by the expression and activity of the non-canonical Robo3.1 receptor . This, tRobo2 mutant embryos’ dI1i neurons. This suggested that within dI1 neurons, the evidence points to the phenotype observed in the Robo2 mutant embryos within the spinal cord as a cell autonomous effect.Here we examine how neurons derived from a common precursor origin respond uniquely to the environment utilizing Robo signaling. A cohort of previous work has demonstrated that embryonic axonal sorting in their longitudinal white matter tracts is influenced by Robo1 and Robo2 . These sRobo1 and Robo2 mRNA and protein broadly correlated in dI1i neurons within the mantle zone, suggesting that in contrast to white matter tracts, the regulatory mechanism within the mantle zone may be at the transcriptional level. Several transcription factors are candidates that could potentially regulate this; however, this is likely to be complex transcriptional regulation and has not been determined (The expression of Robo2 (and Robo1) we observed in dI1 neurons within the mantle zone was substantially weaker than that observed in the latter projecting white matter tracts . This motermined .Robo1 did not statistically significantly enhance the Robo2 mutant dI1i axonal phenotype, suggesting that Robo2 alone was sufficient for the observed axonal phenotype. Of note, while not statistically significant, we observed a marginal mean increase (trend) in axonal misprojections in Robo1/2 double mutants compared with Robo2 mutant embryos. This evidence leaves open the possibility that Robo1 may contribute to the projection of dI1i neurons, which could be for example by contributing to a “threshold” of canonical Robo signaling. We also noted a qualitative but not quantitative dI1i axonal phenotype in Robo1/2 double-heterozygote embryos compared with controls. This modest axonal phenotype Robo1/2 double-heterozygote embryos (two Robo gene copies missing) compared with the strong phenotype in Robo2 mutant embryos (two Robo gene copies missing) suggested that the influence of Robo1 and Robo2 were not equal in dI1i neurons. This suggested that while canonical gene dose may contribute to the guidance, canonical Robos’ influence on dI1i guidance is not mediated by gene dose alone. Taken together, we therefore concluded that Robo2 and Robo1 are not redundant in the context of dI1i axonal projections within the mantle zone and that Robo2 plays the major role. We found a statistically significantly enhanced cell body migration phenotype in E12.5 Robo1/2 double mutant vs. Robo2 mutant embryos, suggesting that in the context of the cell body migration phenotype, Robo1 and Robo2 may be additive. Consistent with our analysis overall, in other contexts, Robo1 and Robo2 can act in a distinct, synergistic, or redundant way depending on the context (We demonstrated that in addition to Robo2, Robo1 was expressed in both dI1i and dI1c neurons, raising the possibility that Robo1 influenced dI1 guidance. We found that loss of  context .This, taken together with other studies, suggests that Robo1 may play a role in commissural axon timing and crossing at the ventral commissure, and here we propose that Robo2 plays a relatively minor role in dI1c neuron guidance within the mantle zone and ventral commissure whereas Robo2 is the major gatekeeper of dI1i guidance.Robo signaling is associated in a wide range of fields including neurodevelopment and neurofunctional disorders in addition to disease processes. Here we have contributed to this body of work demonstrating a neurodevelopmental context in which canonical vs. non-canonical Robos regulate tissue organization. Within the nervous system, understanding how neurons derived from common precursor origin elaborate neural organization has important implications for understanding the assembly of functional connectivity. This study provides an example of how evolution of a gene family permits development of anatomical differentiation/identity from cells derived from a common progenitor pool and initial molecular identity, a concept that is widely applicable throughout different systems and animals. There are a small number of examples of the non-canonical function of Robo receptors in other species . WhetherThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Review Board at the Court of Appeal of Northern Norrland.Robo2 phenotype discovery and preliminary analysis and generated reagents. LG-C designed and performed the Robo1, Robo2 and Robo3 expression quantification, contributed to the migration analysis, characterization of the transgenes and antibodies. CL and CT were involved in an early phase of analysis in the work and generated samples. SW conceived and supervised the work, led the study design, generated the funding, made reagents, performed some experimental work and formal analysis, and wrote the manuscript. All the authors edited, read, and approved the manuscript.MW, MM, LG-C, and CL assembled the figures. MW, MM, and LG-C were responsible for writing parts of the manuscript, experimental design, and performance of experiments. MW performed most of the experiments and analysis in the manuscript and codesigned and performed the phenotype quantifications. MM made the initial 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.


BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.Our previous studies showed an association between monoallelic  BRCA2) gene was identified in 1995 and has been shown to play an essential role in DNA double strand break (DSB) repair by homologous recombination (HR) and DNA crosslink repair by the Fanconi anemia (FA) pathway [BRCA2 gene are known to be highly associated with the risk of diagnosis with breast, ovarian, prostate, and pancreatic cancer [BRCA2 999del5 truncation mutation in exon 9 was isolated in a family with several cases of male and female breast cancer [999del5 mutant and expression studies showed that even though the short mutant RNA is produced there is no detectable corresponding protein, indicating BRCA2 heterozygosity [The breast cancer susceptibility 2 , telomere loss (TL), extrachromosomal telomere sequences (ECTS) and frequent telomere sister chromatid exchanges (T-SCE) as seen in cells that rely on the alternative lengthening of telomeres (ALT) in absence of telomerase. These results indicated the important role of BRCA2 in telomere stabilization and protection [BRCA2 999del5 germline mutation and relative short telomere length measured in blood cells were at significantly higher risk of being diagnosed with breast cancer [BRCA2 gene expression may not be enough to fulfill telomere maintenance, indicating BRCA2 haploinsufficiency. Anticipation effects have also been reported from this study cohort where daughters of mother-daughter pairs with the BRCA2 999del5 germline mutation have been shown to be diagnosed with breast cancer on average 10 years younger than the mothers [Studies on the role of the BRCA2 protein have shown the requirement of HR-dependent RAD51 filament formation plus stabilization by BRCA2 and the core FA pathway for efficient protection in replication forks from degradation following short-term replication arrest. FA and BRCA2 defective cells are defective in fork protection and show an increase in genome instability, manifesting chromatid breaks and radial structures on mitotic chromosome spreads ,16. DNA otection . A recent cancer . These r mothers .BRCA2 999del5 breast cancer cases [BRCA2 wild-type allele in the tumor tissue. Similar results have been reported from another study cohort where the absence of BRCA2 locus-specific loss of heterozygosity was observed in 46% of breast tumors [BRCA2 999del5 breast cancer cases with wild-type allelic loss had significantly worse breast cancer specific survival compared to cases that remained the wild-type allele [BRCA2 haploinsufficient phenotype in vivo in breast tissues of BRCA2 mutation carriers exhibiting DNA damage that resulted from failed RS- and DNA damage responses and consequently aneuploidy [The 50 years old Knudson’s two-hit hypothesis suggests that both alleles of a tumor suppressor gene require to be inactivated to cause tumor formation, as was first described in heritable cases with retinoblastoma . We haveer cases ,22 and per cases  had lostt tumors . Of noteeuploidy .Mechanisms shown to respond to RS that could counteract karyotypic diversity and contribute to tumor progression include polymerase theta-mediated end joining (TMEJ) alternative repair pathway at resected DSBs when HR is deficient to repair broken forks ,27. OtheBRCA2 genotypes including two BRCA2 wild-type cell lines, five BRCA2 heterozygous cell lines with the 999del5 germline mutation and three FA-D1 cell lines with biallelic BRCA2 mutations. The originality of this study was to compare all three BRCA2 genotypes in a cell type not associated with BRCA2 hereditary cancer. Results showed increased telomere abnormalities between the BRCA2 genotypes in a stepwise manner, including TL, ITS and ECTS.In the present study, we compared telomere dysfunction in lymphoid cell lines with different BRCA2 999del5 mutation were Epstein-Barr virus transformed . Telomere fluorescence in situ hybridization (FISH) was performed by ready-to-use Cy3-conjugated PNA pan-telomere probe  in mixture with FITC-conjugated PNA pan-centromere probe (Dako) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) as previously described . The micBRCA2 genotypes were combined and performed as one genotype. A two-tailed Student´s t-test was used for statistical analysis and p < 0.05 was considered statistically significant. The R (CRAN) software was used for graphical representation.Statistical analyses were performed on chromosomal abnormalities as per metaphase and results presented as an average mean with standard error of mean (SEM) bars. Results from each of the At least 60 metaphases were analyzed from each lymphoid cell line with one exception of the HSC62N cell line . Most ofIn addition to analysis of TL, MTS, ITS and ECTS other chromosomal aberrations were measured including sister chromatid and end-to-end chromosome fusions, radial chromosomal configurations and chromosome fragments without telomere signals. These chromosomal aberrations were, however, not included in the results since average events per metaphase were below one which may affect the significance of the results.+/+BRCA2, BRCA2+/- and BRCA2-/- lymphoid cell lines, and a significant difference was found between the BRCA2+/- and BRCA2-/- genotypes  [TL is likely to result from RS that leads to terminal fork collapse, either at a single sister chromatid strand or at both sister chromatids on chromosome ends. TL was found to be increased among us study . In the subgroup . This in signals . Frequenll lines . TelomerCO-FISH) .BRCA2 deficient cells [BRCA2+/+ and the BRCA2+/- and BRCA2-/- lymphoid cell lines, respectively (BRCA2+/- and BRCA2-/- lymphoid cell lines but no difference was, however, found of the total MTS between the two genotypes. Lack of significance in MTS formation between the BRCA2+/- and BRCA2-/- genotypes may be explained by >50% higher TL of the BRCA2-/- genotype (BRCA2+/- and BRCA2-/- genotypes is, however, a strong indication of BRCA2 deficiency related telomere RS due to G-quadruplex formation.MTS is often referred to as fragile telomeres. They have been suggested to represent a recombination event among inter-/intra-chromatid telomeric sequences or ITS in the proximal regions of telomeres  or restant cells . In the ectively . A signigenotype c. The siBRCA2+/- mammary epithelial cell lines compared to a commercial BRCA2+/+ mammary epithelial cell line and cell lines that rely on the classical ALT-positive mechanism for telomere maintenance [BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines (Most ITS probably result from the formation of DSB during RS that is mediated by targeted telomere insertions , althougntenance . In the ll lines  supportiBRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines (BRCA2 genotypes. Sometimes these ECTS have strong fluorescence signals indicating amplification of telomeric c-circles that have been associated with cancer cells that rely on ALT rather than telomerase for telomere maintenance [ECTS found scattered around chromosome spreads presumably result from terminal fork collapse caused by telomere RS. In this study we found a significant stepwise increase of ECTS between the ll lines  probablyntenance . Recent ntenance ,41.BRCA2 genotypes. This was shown in lymphoid cells that are not known to be prone to BRCA2 related cancer risk. It needs to be kept in mind, however, that apart from the BRCA2 genotype the cell lines have generic individual genomic differences. For this same reason, the two BRCA2+/+ control cell lines used in this study may affect the interpretation. The most ideal setup would be to use a homogenous genetic background of a single cell line edited with the CRISPR/Cas9 gene editing technique for insertion of the BRCA2 9999del5 mutation. This has, however, not yet been successful due to the very low viability of cells with the homozygous mutation. This correlates with the fact that no homozygous individuals have been known to exist although expected under the Hardy–Weinberg equilibrium and allele frequency in the Icelandic population [The results clearly show a gradual increase in telomere defects between the BRCA2 dependent loss of HR on Holliday junction resolvases by using HR-intermediates that suppress mitotic crossing over and preserve the genomic stability [BRCA2 deficient cells is not fully understood and needs to be further investigated.Our and other previous findings have shown unequal T-SCE in BRCA2 deficient cells ,18,30. Ctability . BRCA2 dtability ,46,47,48tability . Both RAtability . HoweverBRCA2 999del5 mutation carriers does not differ from non-carriers in blood cells [BRCA2 999del5 mutation carriers was found to be significantly shorter than among non-carriers. Telomere length was shown to be a modifier of breast cancer risk in BRCA2 999del5 mutation carriers in the same study. Another aspect is that BRCA2 999del5 mutation carriers with breast cancer have been associated with a significantly worse prognosis than non-carriers [BRCA2 mutation carriers might be related to consequences of possible telomere RS before and after breast cancer diagnosis needs to be answered in future studies. Such knowledge could have an impact on the prediction of early cancer development, and therefore, be used to advise the timing of preventive therapies, and targeted therapies of BRCA2 related breast cancer in the future as discussed here above.Telomere length homeostasis in unaffected od cells . After acarriers . How defBRCA2+/+, BRCA2+/- and BRCA2-/- genotypes showing a strong indication of BRCA2 haploinsufficiency in telomere maintenance. Our results from telomere FISH show a clear increase in TL, MTS, ITS and ECTS between the genotypes that are a sign of telomere RS in BRCA2 deficient cells. More research is needed on the possible involvement of the RAD52 related BIR and the POLQ related TMEJ backup pathways in telomere maintenance when BRCA2 is not fully expressed and the distinct roles of the two pathways on telomere maintenance and genomic stability.Taken together, we found a stepwise increase in telomere abnormalities between the


FOXF2, encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. FOXF2 is located in a conserved gene cluster containing FOXQ1, FOXF2, and FOXC1. We found that expression of Foxq1 is dramatically upregulated in the embryonic palatal mesenchyme in Foxf2–/– mouse embryos. We show here that the Foxf2 promoter-deletion mutation caused dramatically increased expression of the cis-linked Foxq1 allele but had little effect on the Foxq1 allele in trans. We analyzed effects of the Foxf2 mutation on the expression of other neighboring genes and compared those effects with the chromatin domain structure and recently identified enhancer-promoter associations as well as H3K27ac ChIP-seq data. We show that the Foxf2 mutation resulted in significantly increased expression of the Foxq1 and Exoc2 genes located in the same topologically associated domain with Foxf2 but not the expression of the Foxc1 and Gmds genes located in the adjacent chromatin domain. We inactivated the Foxq1 gene in mice homozygous for a Foxf2 conditional allele using CRISPR genome editing and generated +/–(Foxf2/Foxq1) mice with loss-of-function mutations in Foxf2 and Foxq1 in cis. Whereas the –/–(Foxf2/Foxq1) mice exhibited cleft palate at birth similar as in the Foxf2–/– mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (Foxf2/–/–Foxq1) embryos exhibited distinct effects on the domain-specific expression of several important genes, including Foxf1, Shox2, and Spon1, in the developing palatal shelves compared with Foxf2–/– embryos. These results identify a novel cis-regulatory effect of the Foxf2 mutation and demonstrate that cis-regulation of Foxq1 contributed to alterations in palatal gene expression in Foxf2–/– embryos. These results have important implications for interpretation of results and mechanisms from studies of promoter- or gene-deletion alleles. In addition, the unique mouse lines generated in this study provide a valuable resource for understanding the cross-regulation and combinatorial functions of the Foxf2 and Foxq1 genes in development and disease.Disruption of  The secondary palate separates the nasal cavity from the oral cavity and consists of the bony hard palate anteriorly and muscular soft palate posteriorly . In mammShh mRNAs to the oral side palatal epithelium to control the oral-nasal patterning  and chromatin immunoprecipitation-sequencing (ChIP-seq) mediated genome wide mapping of Foxf2 binding sites to identify direct Foxf2 target genes in the developing palatal mesenchyme , 2020. If growth , we showf growth . Among t embryos . Foxq1 i embryos . Foxq1–/reported . Remarka genomes . In bothch other . Thus, t editing  and our Foxf2tm1Rhc (Foxf2c/c) and Foxf2+/– mice have been described previously  (50 ng/μl each) targeting the genomic sequence flanking the Forkhead domain-coding sequence in the Foxq1 gene were co-injected with humanized Cas9 mRNAs (50 ng/μl) into zygotes of Foxf2c/c mice  were used in this study. Founder mice were crossed to Foxf2c/c mice to generate the Foxf2c/c; Foxq1+/– mice. Genotypically verified G1 Foxf2c/c; Foxq1+/– mice were intercrossed to generate Foxf2c/c; Foxq1–/– homozygotes. In addition, Foxf2c/c; Foxq1+/– mice were crossed to EIIa-Cre transgenic mice  mice. +/–(Foxf2/Foxq1) mice were intercrossed to generate –/–(Foxf2/Foxq1) homozygous embryos for analyses. For timed mating, noon of the day on which a vaginal plug was identified was designated as embryonic day (E) 0.5. All animal work procedures were performed following recommendations in the Guide for Care and Use of Laboratory Animals by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children’s Hospital Medical Center. This study conformed with the ARRIVE  guidelines for preclinical animal studies.eviously  and werec/c mice . The tarnic mice  to inactin situ hybridization as described previously  of the Foxq1 gene (rs29587452) between the C57BL/6J and 129X1/SvJ mouse strains. Purified PCR products amplified from wildtype, +/–Foxf2, and Foxf2–/– cDNA samples were digested overnight using AciI  at 37oC. Quantification of the digested and undigested DNA fragments was performed by QIAxcel Advanced using QIAxcel® ScreenGel software .Whole palatal shelves of E13.5 embryos were manually dissected in ice-cold phosphate buffered saline. Total RNAs were extracted using the RNeasy micro kit . First-strand cDNAs were prepared using the SuperScript III First-Strand Synthesis System , and real-time qPCR was performed using a CFX96 Real-Time System  with conditions recommended by the manufacturer. Relative levels of mRNAs in each sample were normalized to that of t test was used for pairwise comparison. One-way ANOVA followed by Newman–Keuls post hoc test was used to compare all pairs when more than two genotypes were included. P < 0.05 was considered significantly different.For statistical analysis, all results were presented as mean ± SEM. Student’s 1 (2 (accession number GSE138721) (3 (The whole genome chromosome conformation capture (Hi-C) data and topologically associated domain (TAD) map of mouse embryonic stem cells were retrieved from the 3D Genome Browser1 . The ori1 , and assE138721) . The rep8721)  gene, named 1700018A04Rik, which is transcribed from the opposite DNA strand with the most 5′ transcription start site (TSS) located only about 300 bp from the TSS of Foxf2  that is disrupted in the 129SvEv allele. We designed a pairs of PCR primers flanking SNP rs29587452 and used AciI restriction fragment polymorphism to analyze possible differential expression of the two Foxq1 alleles in the Foxf2+/– mutant palate shelves  and 494  bp, respectively, spanning the entire Forkhead domain-coding region of the Foxq1 gene were established and used in this study +/– progeny to C57BL/6J mice to establish the +/–(Foxf2/Foxq1) mouse colony. These +/–(Foxf2/Foxq1) mice allowed us to further investigate allele-specific effects of the Foxf2 mutation on the linked Foxq1 allele by direct quantitative comparison of allele-specific Foxq1 expression between different embryos of distinct genotypes  embryos in comparison with their wildtype littermates. However, the amount of mRNAs produced from the wildtype Foxq1 allele in the +/–(Foxf2/Foxq1) embryos was only about 50% of the amount of Foxq1 mRNAs in the wildtype sample  studies have demonstrated that mammalian genomes are organized into a series of topologically associated domains (TADs), megabase-scale genomic intervals where interactions between enhancers and gene promoters take place more frequently within than across adjacent TADs . Althougcent TAD . RT-qPCRtermates . Expressd either . On the termates . In situ embryos . Analysi embryos  indicatend Foxq1 . Furtherpromoter . Remarkapromoter  showed t tissues . In addi tissues  revealed tissues . Togethe+/–(Foxf2/Foxq1) mice generated –/–(Foxf2/Foxq1) mutant mice with over 80% of the homozygous mutants exhibited complete cleft palate (–/–(Foxf2/Foxq1) embryos at multiple developmental stages by histology or skeletal preparations revealed that craniofacial anomalies, including cleft palate, were similar as the phenotypes of Foxf2–/– embryos described previously  embryos displayed defects in palatal shelf elevation  embryos as previously reported for Foxf2–/– mutant embryos  embryos in comparison with wildtype littermates at E13.5  embryos compared with the wildtype littermates  mutant embryos  embryos in comparison with the wildtype littermates by both whole mount in situ hybridization and real-time RT-qPCR assays  mutant embryos  embryos (–/–(Foxf2/Foxq1) embryos in comparison with the wildtype embryos . Interestingly, we also consistently detected moderately increased expression of Foxf1 protein in the posterior region of the palatal shelves in Foxf2–/– embryos compared with the wildtype embryos (H with G). These results indicate that Foxf2 negatively regulates Foxf1 gene expression in the developing palatal mesenchyme and that the increased expression of Foxq1 in the Foxf2–/– embryonic palatal mesenchyme partly complemented for Foxf2-mediated regulation of Foxf1 expression. Altogether, although deletion of Foxq1 was ultimately insufficient to rescue the cleft palate defects in the Foxf2–/– mice, the increased expression of Foxq1 resulting from the cis-regulatory effect of the Foxf2 mutation affected the regulation of multiple important genes in palate development.We previously generated and analyzed elopment . Anothern assays . Our preR assays . In the at E13.5 . The exp embryos . We furt embryos . The FoxFOXF2 is located in an evolutionarily conserved gene cluster containing FOXQ1, FOXF2, and FOXC1  mice carrying null alleles of Foxf2 and Foxq1 in cis, and identified a novel cis-regulation of Foxq1 expression by the Foxf2 mutation during palate development. Our results indicate that Foxq1 affected expression of several previously identified Foxf2-dependent genes in palate development, which needs to be taken into consideration for elucidating the molecular mechanisms involving Foxf2 in regulating palate development.nd FOXC1 . Disruptn humans , making in vitro study suggested that FOXF2 binds to the promoter and represses the expression of FOXQ1 in a breast cancer cell line  embryos. This is possibly due to the relatively low level of expression of Foxq1 in the Foxf2–/– palatal mesenchyme compared with the amount of Foxf2 mRNAs expressed in the wildtype palatal mesenchyme. On the other hand, previous studies have suggested that FOXF2 and FOXQ1 play opposite roles in controlling epithelial-mesenchymal transition and visceral metastasis in basal-like breast cancer cells  embryos compared with the Foxf2–/– and wildtype control embryos  embryos  embryos, the increased expression of Foxf1 was insufficient to rescue the cleft palate phenotype or the altered expression of many genes in the posterior palatal mesenchyme that resulted from Foxf2 disruption, suggesting that Foxf2 has distinct molecular functions in palate development that could not be complemented by either Foxq1 or Foxf1.The expression of er cells . Other ser cells . We foun embryos . While t embryos . In thisFoxf2 gene promoter had a cis-regulatory effect on the expression of nearby genes. Our results clearly demonstrate that the increased expression of Foxq1 in the Foxf2+/– and Foxf2–/– embryos was due to the cis-regulatory effect. Although –/–(Foxf2/Foxq1) mice exhibited similar cleft palate phenotypes as Foxf2–/– mice, Foxq1 exerted a regulatory effect on the expression of several previously identified Foxf2-dependent genes in palate development. We show that Exoc2, located about 600 kb upstream of Foxf2, was also significantly upregulated in the developing palatal shelves in Foxf2+/– embryos and was further significantly upregulated in the Foxf2–/– embryos. Whether the increase in Exoc2 expression in the palatal tissues in Foxf2+/– and Foxf2–/– embryos was solely due to the cis-regulatory effect and whether the altered expression of Exoc2 contributed significantly to the craniofacial and other developmental defects in the Foxf2–/– mice remain to be investigated. Since many gene knockout studies assigned gene function based on promoter-deletion alleles, our finding of a cis-regulatory effect of the Foxf2 mutation calls for caution in interpretation of results and underlying mechanisms from those alleles. Potential cis-regulatory effects on neighboring genes should also be taken into consideration when analyzing pathogenicity of human gene deletion variants. In addition, since Foxf2 mutant mouse studies have shown that multiple tissues and developmental processes, including pericyte development and maturation of the blood-brain barrier, development of the respiratory and digestive organs, in addition to craniofacial and palate tissues, depend on Foxf2 function (reviewed by +/–Foxf2/Foxq1) mice provide a valuable resource for understanding the cross-regulation and combinatorial functions of the Foxf2 and Foxq1 genes in multiple developmental and disease processes.In summary, this study demonstrates that disruption of the GSE67015. The accession number for the palate Foxf2 ChIP-seq data is GSE137585.Publicly available datasets were analyzed in this study. This data can be found here: The accession number for the RNA-seq data is The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children’s Hospital Medical Center.YL and RJ designed the research. JX and HL conducted the experiments and collected the data. JX and RJ wrote the manuscript. All authors analyzed the data, revised the manuscript, and agreed and approved the manuscript.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.


However, there is no consensus with respect to the responsiveness of these O2 resaturation parameters for assessing reactive hyperemia.There is a spectrum of possibilities for analyzing muscle O2 resaturation parameters to assess reactive hyperemia in the microvasculature of a clinical group known to exhibit impairments of tissue O2 saturation (StO2).This study investigates the responsiveness of the most utilized muscle O2 after a 5-min arterial occlusion challenge and the following parameters were analyzed: StO2slope_10s, StO2slope_30s, and StO2slope_until_baseline ; time to StO2baseline and time to StO2max ; ∆StO2reperfusion ; total area under the curve (StO2AUCt); and AUC above the baseline value (StO2AUC_above_base).Twenty-three healthy young adults, twenty-nine healthy older adults, and thirty-five older adults at risk of cardiovascular disease (CVD) were recruited. Near-infrared spectroscopy (NIRS) was used to assess StO2slope_10s was significantly slower in older adults at risk for CVD compared to healthy young individuals (p < 0.001) and to healthy older adults (p < 0.001). Conversely, time to StO2max was significantly longer in healthy young individuals than in older adult at CVD risk.Only StO2slope_10s may be a measure of reactive hyperemia, which provides clinical insight into microvascular function assessment.Our findings suggest that StO Since NIRS detects changes in oxygenated and deoxygenated hemoglobin in the tissues using absorption of near-infrared light at a specific wavelength,,,Over the past decades, near-infrared spectroscopy (NIRS) combined with a vascular occlusion test (VOT) has been used for assessment of tissue O2 signal follows a downward path during the cuff inflation phase and then StO2 goes rapidly upward immediately after cuff deflation (reperfusion phase).-2 signal during reperfusion enables various StO2 parameters to be calculated, which, in general, are interpreted as measures of microvascular function.2 parameters adopted in previous studies include reperfusion rate ,-2 and the difference between the lowest and highest StO2 value),,-2 baseline, time to StO2 maximum, and AUC above the baseline).,,,Performing a VOT with an NIRS device fitted to the limb of interest, the StO2 resaturation parameters, the lack of standardization of which NIRS parameters are used during the reperfusion phase makes comparisons between studies difficult and/or may even lead to misinterpretation of results. For example, since the time to maximum StO2 parameter is calculated as the time elapsed from release of the cuff to the maximum StO2 value during reperfusion, individuals with higher O2 extraction capacity  might exhibit a longer time to maximum StO2. In other words, in individuals with higher O2 extraction, the distance between the lowest StO2 value reached during cuff inflation and the highest StO2 reached during the cuff deflation is longer, thereby influencing calculation of some NIRS parameters, such as time to StO2 maximum and even area under the reperfusion curve. Therefore, the present study investigates the usefulness of the muscle O2 resaturation parameters most utilized to assess reactive hyperemia in a clinical group known to exhibit impairments in muscle StO2. It was hypothesized that some NIRS parameters might be more sensitive than others (mainly those influenced by muscle O2 extraction) for detecting abnormal reactive hyperemia in a clinical sample.Despite a range of possibilities for interpretation of muscle O-three healthy young individuals, 29 healthy older adults, and 35 older adults at risk of CVD were recruited to participate in the study. For the younger group, inclusion criteria were being healthy (not presenting CVD risk factors) and age < 35 years. For the healthy older adult group, individuals had to be healthy and ≥ 60 years of age. The inclusion criteria for older adults at high risk for CVD were age ≥ 60 years and possessing at least four CVD risk factors, according to a previous study demonstrating that presence of three or more CVD risk factors is related to microvascular dysfunction.,o. 1.489.668).TwentyAfter 12 h fasting, the participants arrived at the laboratory, where they underwent anthropometric measurement  before blood samples were taken. Blood samples were collected only at baseline in order to determine the CVD risk factors  of the participants enrolled on the study. Afterwards, participants rested for 10 min in the supine position on an examination table, followed by assessment of the NIRS parameters for level of muscle oxygen saturation. The experimental procedures were conducted between 8:00 and 10:00 a.m. and participants were recommended to avoid caffeine and foods rich in nitrates and nitrites for 24 h before the laboratory visit.2 saturation (%StO2) was assessed using an NIRS system  connected to a personal computer via Bluetooth for data acquisition (10 Hz). Analogue‐to‐digital conversion and subsequent analysis of the raw data  was conducted using native software , as previously described by Oliveira et al.,2 was recorded continuously throughout the VOT . The cuff was placed over the arm, 2 cm above the medial epicondyle of the humerus and a cuff pressure of 250 mm Hg was used during the occlusion period  StO2 during baseline  was calculated as the average over the 30 s before cuff occlusion; (ii) the lowest StO2 value reached during cuff inflation (StO2min) and the highest StO2 value reached following cuff deflation (StO2max); (iii) time for the StO2 signal to reach its peak after cuff release (time to StO2max); (iv) time for the StO2 signal to reach the pre-occlusion baseline value after cuff release (time to StO2base); (v) difference between StO2min and the StO2max after cuff deflation (ΔStO2reperfusion); (vi) the area under the reperfusion curve (AUC) above the baseline (StO2AUCabove_base); (vii) the total AUC over 2 minutes of reperfusion (StO2AUCt); (viii) upslope of StO2 signal over a 10 s reperfusion window immediately following cuff deflation (StO2slope_10s); (ix) upslope of StO2 signal over a 30 s reperfusion window immediately following cuff deflation (StO2slope_30s); and (x) upslope of StO2 signal over the time elapsed between StO2min and reaching pre-occlusion baseline StO2 values  for a specific test . Based on a statistical power (1 – β) of 0.80, an effect size of 0.46, and an overall significance level of 0.05, 51 participants would be needed to detect a statistical difference. The effect size was based on a previous study that found d=0.93  when assessing reperfusion rates in older individuals and young participants.One-way ANOVA was used to identify significant differences between the characteristics of the participants at baseline and tissue StOBaseline characteristics of the participants and medications used are shown in 2 saturation parameters are shown in 2slope_10s (P < 0.001), and StO2slope_30s (P < 0.001), and blunted ΔStO2reperfusion (P < 0.001), StO2AUCabove_base (P < 0.001), StO2AUCt (P < 0.001), StO2max (P = 0.035), and StO2 min (P < 0.001) were observed in older adults at CVD risk compared to healthy young individuals. Additionally, StO2slope_10s was significantly slower (P < 0.001) in older adults at risk for CVD compared to their healthy counterparts. Time to StO2max was significantly slower (P = 0.047) in healthy young individuals than older adults at risk of CVD.Muscle StO2slope_30s (P = 0.003) and blunted ΔStO2reperfusion (P < 0.001), StO2AUCabove_base (P = 0.006), StO2AUCt (P = 0.001), and StO2 min (P = 0.002) were found in healthy older adults compared to healthy young people. No significant differences in StO2base, time to StO2base, or StO2slope_until_base were found between groups (P > 0.05).Significant slower StO2 resaturation parameters as a measurement of reactive hyperemia in the microvasculature of different populations, the present study investigated the effectiveness of the O2 resaturation parameters most frequently used for evaluating reactive hyperemia in healthy older adults or older adults with CVD risk factors. It was hypothesized that analyzing the effectiveness of the most widespread NIRS parameters for detecting aging and CVD risk factors might help with development of criteria for establishing an appropriate set of muscle StO2 parameters in order to facilitate the choice between different methodologies of NIRS analysis.Given the widespread utilization of NIRS devices and the spectrum of possibilities for analyzing muscle O2 resaturation rate parameters  are largely adopted to assess reactive hyperemia in hypertensive individuals,,,,2slope_10s was significantly slower in older adults with CVD risk factors compared to healthy older adults and to healthy young individuals, suggesting that StO2slope_10s may be a more sensitive NIRS parameter for assessing reactive hyperemia. The StO2slope_10s has typically been adopted to assess O2 resaturation rate, since previous studies have reported that this parameter is correlated with reactive hyperemia measured in the brachial artery,2slope_10s may be a stimulus for FMD response given that FMD is evoked by increasing blood flow after cuff release.,,2slope_10s may be considered an appropriate way to analyze O2 reperfusion rate given the linearity of the StO2 signal over the initial 10 s after cuff release.Muscle O2max may be mainly affected by the degree of muscle O2 extraction (StO2min), since this recovery time parameter measures the time interval between the StO2min  and StO2max . Therefore, individuals with higher muscle O2 extraction capacity  will reach a lower StO2 value during the cuff inflation phase (StO2min), which makes the time to StO2max longer for those individuals with higher O2 extraction capacity.,2max was found in healthy individuals compared to older adults at CVD risk, which may have been the result of a lower StO2 value reached during cuff inflation (StO2min), combined with the higher StO2 value reached during the cuff deflation phase (StO2max).Time to StO2max in individuals with metabolic syndrome2max in individuals with peripheral vascular disease  compared to healthy participants. Interestingly, the groups investigated in their study2 extraction parameters, which may explain the expected longer time to StO2max in individuals with peripheral vascular disease. Therefore, adoption of the recovery time parameters  should be recommended when the clinical population evaluated is not assumed to exhibit impairment in muscle O2 extraction; otherwise, data interpretation and comparison may be rendered incorrect.Previous studies have failed to show significant differences in time to StO2reperfusion and StO2AUCt in healthy older adults and those with CVD risk factors, compared to healthy young individuals. However, no significant difference was observed in these parameters in healthy older adults compared to older adults at risk for CVD, suggesting that ΔStO2reperfusion and StO2AUCt are not sensitive parameters for detecting the effect of CVD risk factors on reactive hyperemia. The ΔStO2reperfusion and StO2AUCt parameters can be interpreted as measures of the magnitude of O2 resaturation, since the lowest and highest StO2 value (amplitude) within the reperfusion phase (cuff deflation) are considered for calculating these parameters. However, it is important to note that, in common with time to StO2max, these parameters may also be affected by parameters of muscle O2 extraction during cuff deflation (i.e. StO2min).The current study also demonstrated a significant reduction in ΔStO2AUCt observed in older adults at CVD risk in comparison to healthy young individuals was eliminated when the level of muscle O2 extraction (StO2min) was standardized by varying occlusion times  across the two age groups. It is likely that differences between ΔStO2reperfusion and StO2AUCt parameters do not only necessarily reflect impairment in O2 resaturation magnitude (reactive hyperemia), but also in muscle O2 extraction during occlusion .,2 saturation throughout the VOT, assessment of ΔStO2reperfusion and StO2AUCt could be performed controlling the level of tissue ischemia (varying the occlusion times),2reperfusion and StO2AUCt. Alternatively, AUCabove_base is not affected by muscle StO2min and its utilization might be recommendable.Corroborating this idea, Rosenberry et al.2 saturation. For example, a previous study conducted at our laboratory showed that prolonged occlusion of limb blood flow  causes transient microvascular dysfunction that can be assessed by NIRS‐derived reperfusion upslope.,,Of note, the NIRS system is a relevant tool that can be easily used during vascular surgery, as it continuously assesses muscle O,,,,Although NIRS systems are largely utilized to assess oxygenation of the forearm muscles, one limitation of this technique is the discrete volume of a specific muscle (2 to 6 cm) and the superficial area of skeletal muscle that NIRS examines.2slope_10s was the most sensitive NIRS parameter for assessing reactive hyperemia. Additionally, the magnitude of reperfusion  and recovery time  parameters should be interpreted with caution when assessing clinical populations, since muscle O2 extraction may affect those parameters. The results of the present study will be helpful for future studies that investigate the effect of an external factor  on muscle O2 resaturation parameters.In conclusion, the findings of this study suggest that StO


Transesophageal echocardiography (TEE) and multidetector computed tomography imaging revealed type 0 calcified bicuspid AV based on the Sievers classification   with an aortic valve (AV) area of 0.74 cm2  small curve wire in the mid LV, balloon aortic valvuloplasty (BAV) was tried using Z-MEDII 23 × 40 mm  to make it easier to cross the device due to very severe AS and to confirm the valve size in type 0 bicuspid AV. However, the balloon could not cross to AV because of strong resistance at the ascending part to the arch graft and the tortuous thoracoabdominal aorta  and advanced another stiff guidewire through a 5 French pigtail catheter  at the ascending part to the arch graft and the tortuous thoracoabdominal aorta.1 (MP4 7148 KB)Online video 2: Easy crossing of SAPIEN 3 through the aorta and aortic valve.2 (MP4 9670 KB)Below is the link to the electronic supplementary material.


This 3-phase study involves the conceptualization and design, development and usability testing of a Comprehensive Digital Self-care Support System (CDSSS) named myHESTIA for older adults with multiple chronic conditions (MCC). The objective of this study was to test whether a CDSSS can be developed for those who are dealing with MCC and whether such a system that is specifically developed for older adult patients will enable daily capture of self-care data. Participants for this 3-phase study included: 10 older adults (age>60) and 10 caregivers in Phase 1; 15 Geriatrics clinicians and 25 community-dwelling low-income older adults in Phase 2; and, 10 older adults (age>60) with MCC in Phase 3. Agile method of system development was used for the design and development of the system. The first two phases involved collecting data for designing and developing myHESTIA. The third phase involved small group usability and feasibility testing, in which the participants used myHESTIA trackers for 4 weeks. Results from phase 3 shows daily inputs were possible and the self-reported data shows that it was not at all difficult for older adults to track their symptoms daily. User experience data (n=10) shows overall positive experience along pragmatic (5.8 out of 7), hedonic (4.6 out of 7), sociability (5.5 out of 7) and usability (6.3 out of 7) experience dimensions. Finally, all the participants (n=10) who completed the phase 3 study reported intention to continue using myHESTIA. Results indicate that it is feasible to design a CDSSS for older adults with MCC.