Their physicochemical and mechanical properties were then determined. Both MMT and PLG nanoclays were added into monomer mixture (1:1 ratio) at different loading levels , and the resulting composites were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), dynamic mechanical analysis (DMA) and mechanical testing (bending and compressive properties). Thermal properties, depth of cure and water absorption were not greatly affected by the type of nanoclay, while the mechanical properties of dental resin composites depended on both the variety and concentration of nanoclay. In this regard, composites containing MMT displayed higher mechanical strength  than those resins prepared with PLG due to a poor nanoclay dispersion as revealed by SEM. Solubility of the composites was dependent not only on nanoclay-type but also the mineral concentration. Dental composites fulfilled the minimum depth cure and solubility criteria set by the ISO 4049 standard. In contrast, the minimum bending strength (50 MPa) established by the international standard was only satisfied by the dental resins containing MMT. Based on these results, composites containing either MMT or PLG (at low filler contents) are potentially suitable for use in dental restorative resins, although those prepared with MMT displayed better results.Bis-GMA/TTEGDMA-based resin composites were prepared with two different types of nanoclays: an organically modified laminar clay (Cloisite Dental resin-based composites (RBCs) for both direct and indirect dental restorations have been in use for the past 50 years . These mBisphenol A glycidyl methacrylate (Bis-GMA) is the base-monomer most frequently used in the formulations of dental restorative materials since it reduces polymerization shrinkage and enhances both modulus and thermal stability of the resulting materials; however, it also exhibits a high viscosity, which yields usually a heterogeneous material and problems during handling and application of the product. Thus, in order to achieve high filler loading in dental resin composites, low-viscosity diluent monomers such as triethylene glycol dimethacrylate (TEGDMA) are commonly used ,4,5. OthMonomers used in the commercial formulations of dental composites have remained largely unchanged, whereas the type, shape, size and distribution of inorganic fillers have undergone significant changes . RecentlIt is worth mentioning that these improvements are generally achieved through addition of a small amount of nanoclay . This fact is due to the extremely large interface area provided by the nano-size particles of the suspended filler .® Na+ [® 10A [® 20A [® 93A [® 30B is the organoclay most employed [At present, there is an increasing interest in the incorporation of nanoclays into dental composites. Montmorillonite (MMT) is the most commonly used clay, both in its natural form and organically modified. Its use has been reported in Cloisite® Na+ ,7,9; CloA [® 20A  and Cloiemployed ,6,7,11.In contrast, there are few studies in the literature involving the use of alternative clay minerals. Tian et al. and Zhang et al. reported the used of palygorskite (attapulgite) in dental resins, while Weidenbach et al. studied materials containing functional halloysite–nanotube filler ,13,14.Therefore, the aim of this work is to investigate the influence of nanoclay type at different loading levels  on properties of the dental resins composites. Tetraethylene glycol dimethacrylate (TTEGDMA) was used as co-monomer instead of triethylene glycol dimethacrylate (TEGDMA) monomer, as Rüttermann et al. suggested that viscosities of both monomers are similar but the molecular mass of TTEGDMA is higher than TEGDMA; in consequence, it was expected to be more advantageous regarding shrinkage . The TTEN,N-dimethyl aminoethyl methacrylate (DMAEMA) were used as photo-initiator and co-initiator, respectively. All reagents were purchased from Sigma–Aldrich Co.  and used as received without further purification. The clay minerals used in this study were a commercial montmorillonite  modified with a quaternary ammonium salt (90 meq/100 g of clay) from Southern Clay Products  and an HCl purified palygorskite (PLG) extracted from a mineral deposit in Chapab, Yucatán, México.The monomers used for the preparation of the dental composites were Bisphenol A glycidyl methacrylate (Bis-GMA) and tetraethylene glycol dimethacrylate (TTEGDMA). Camphorquinone (CQ) and 2. Bis-GMA was mixed manually with TTEGDMA in a glass container at 50:50 wt.% ratio. The photo-initiator CQ and the tertiary amine DMAEMA were then added to the Bis-GMA/TTEGDMA mixture . The container was covered with aluminum foil (to avoid premature curing) and refrigerated until its use. Monomers and initiator system were manually mixed, and the nanoclay (MMT or PLG) was slowly added at different loading levels in small portions to avoid the formation of agglomerates; formulations were mixed until no filler agglomerations were visually observed in the monomer mixture. Resin composites were cured with a light emitting diode unit  with a wavelength range of 420–480 nm and a light intensity of 1000 mW/cm−1 wavenumber range with a resolution of 4 cm−1 and averaging 100 scans.Infrared spectra were obtained with a Thermo Scientific Nicolet 8700 spectrometer  by the attenuated total reflectance (ATR) technique using a germanium crystal. Samples were analyzed in the 4000 to 650 cmThermogravimetric analysis was performed in a Perkin Elmer TGA-7 thermogravimetric analyzer , from 45 to 750 °C at a heating rate of 10 °C/min, under nitrogen atmosphere. From the first derivative curve, the decomposition temperature of composites was determined.The glass transition temperature (Tg) of composites was determined by dynamic mechanical analysis using a Perkin Elmer DMA-7  in bending mode. Bars of 30 × 10 × 0.5 mm were heated from 35 to 200 °C at 3 °C/min, under nitrogen atmosphere, using a frequency of 1 Hz.An overlapping irradiation regime was applied to photo-polymerize the specimens for mechanical properties dependent on size and shape; five overlapped irradiations were employed on both sizes  to cure bending specimens, while one irradiation was used on both sizes  in compression specimens. All photo-cured specimens were immersed in distilled water at 37 ± 1 °C until mechanical testing (7 days).In order to establish the influence of both nanoclay-type and filler content on the mechanical properties of dental resins, flexural tests were carried out according to the ISO 4049 standard  while coFlexural strength  using a JEOL JSM-6360 LV  operated at 20 kV. Samples were adhered on aluminum cylinders using a double-sided tape of copper and coated with a thin layer of gold in a Denton Vacuum Desk-II  sputter coater system for 1 min prior to examination.Depth of cure  tests were performed on cylindrical specimens  according to Section 7.10 of the ISO 4049 standard . Three s3) and solubility  were calculated according to Equations (5) and (6), respectively.For water sorption and solubility tests, disc-shaped specimens  were used according to Section 7.12 of the ISO 4049 standard . Nine ovOne-way analysis of variance (ANOVA) and Tukey’s test (P < 0.05) were used to determine significant differences between properties of dental resin composites prepared with either MMT or PLG.−1, attributed to O-H stretching vibration of hydroxyls in Bis-GMA structure; bands at 2951 and 2877 cm−1 related to asymmetric and symmetric stretching vibrations of the methylene group, and an intense band at 1723 cm−1 owing to C=O stretching of ester groups from dimethacrylates (Bis-GMA and TTEGDMA). Spectra also showed bands at 1640 and 1608 cm−1 which correspond to the stretching vibration of the aliphatic (from vinyl group of monomers) and aromatic (from benzene ring) C=C bonds, respectively [−1 was also detected and associated with symmetric vibration of C-O-C linkage from TTEGDMA structure.FTIR spectra of neat resin and its nanocomposites prepared with either MMT or PLG are shown in ectively ; in factectively . FinallyOn the other hand, it has been suggested that addition of nanoclays into polymeric matrices could improve the thermal stability of the resulting material . This faFinally, it should be also mentioned that the broad transition observed at 320 °C was shifted to lower temperatures (290 °C) in nanocomposites, being more evident when nanoclay content was increased. This event could be related to the emission of volatiles such as water and fragments of organic modifier from PLG and MMT nanoclays respectively.Glass transition temperatures of dental composites were obtained from the maximum of the tan d versus temperature curves and their maxima are summarized in Regarding flexural properties , it was The flexural modulus tended to increase with filler concentration regardless of type of nanoclay employed; MMT composites seemed to exhibit slight increments, but the differences were not statistically significant). PGL composites showed higher modulus with PLG up to 4 wt.% clay content, and no statistical differences were found for composites with higher clay content. Several authors ,25 have It is well known that the dispersion of nanoparticles and compatibility between phases (filler and matrix) play a key role in the enhancement of mechanical properties of nanocomposites. Thus, it is probable that MMT presented better results than PLG as it was organically modified to improve its interaction with organic polymers and increase the interlayer spacing favoring its dispersion, as observed in the SEM analysis see .Compressive properties  were notHigher curing depth of restorative materials is one of the factors that determine the quality of the material . Curing The water sorption for neat resin and its composites prepared with either MMT or PLG at different contents are shown in 3 , whereas all nanocomposites exhibited water sorption values slightly higher.The hydrophilic behavior of composites depends on characteristics of constituents, i.e., organic matrix and inorganic filler. In this regard, unfilled resin exhibited a water absorption slightly below 40 mg/mmInterestingly, composites containing PLG exhibited a similar water sorption behavior to that displayed by materials prepared with MMT, although the latter clay was modified organically by a cation exchange reaction between the silicate and methyl, tallow, bis-2-hydroxyethyl and quaternary ammonium chloride in order to reduce the clay hydrophilicity. This could be attributed to the presence of two hydroxyethyl groups in the organoclay as suggested by Mucci et al. .Water uptake in dental resin composites occurs by diffusion of water molecules within a polymeric matrix and may cause hydrolytic degradation of the matrix and/or filler matrix interface , yieldin3 as suggested by the ISO 4049 [ISO 4049  standardBis-GMA/TTEGDMA-based resin composites with two different types of nanoclays were successfully prepared and characterized. Results indicate that Tg, Td, depth of cure and water absorption were not greatly affected by the type of nanoclay, while the mechanical properties of dental resin composites depended on nanoclay type and concentration of inorganic filler. In general, MMT composites displayed higher mechanical strength than those shown by resins prepared with PLG, due to dispersion problems as revealed by SEM. Solubility of the composites was also dependent on nanoclay type and the mineral concentration. In general, dental composites prepared in this study fulfilled the minimum depth cure and solubility criteria set by the ISO 4049 standard. In contrast, the minimum bending strength (50 MPa) established by the international standard was only satisfied by dental resins containing MMT. Based on these results, composites containing either MMT or PLG (at low filler contents) are potentially suitable for use in dental restorative resins, although those prepared with MMT displayed better results.


Despite obvious advances over the last decades, locally advanced adenocarcinomas of the gastroesophageal junction (GEJ) still carry a dismal prognosis with overall 5-year survival rates of less than 50% even when using modern optimized treatment protocols such as perioperative chemotherapy based on the FLOT regimen or radiochemotherapy. Therefore the question remains whether neoadjuvant chemotherapy or neoadjuvant radiochemotherapy is eliciting the best results in patients with GEJ cancer. Hence, an adequately powered multicentre trial comparing both therapeutic strategies is clearly warranted.The RACE trial is a an investigator initiated multicenter, prospective, randomized, stratified phase III clinical trial and seeks to investigate the role of preoperative induction chemotherapy  with subsequent preoperative radiochemotherapy  compared to preoperative chemotherapy alone (4 cycles of FLOT), both followed by resection and postoperative completion of chemotherapy (4 cycles of FLOT), in the treatment of locally advanced, potentially resectable adenocarcinoma of the gastroesophageal junction. Patients with cT3–4, any N, M0 or cT2 N+, M0 adenocarcinoma of the GEJ are eligible for inclusion. The RACE trial aims to enrol 340 patients to be allocated to both treatment arms in a 1:1 ratio stratified by tumour site. The primary endpoint of the trial is progression-free survival assessed with follow-up of maximum 60 months. Secondary endpoints include overall survival, R0 resection rate, number of harvested lymph nodes, site of tumour relapse, perioperative morbidity and mortality, safety and toxicity and quality of life.The RACE trial compares induction chemotherapy with FLOT followed by preoperative oxaliplatin and 5-Fluorouracil-based chemoradiation versus preoperative chemotherapy with FLOT alone, both followed by surgery and postoperative completion of FLOT chemotherapy in the treatment of locally advanced, non-metastatic adenocarcinoma of the GEJ. The trial aims to show superiority of the combined chemotherapy/radiochemotherapy treatment, assessed by progression-free survival, over perioperative chemotherapy alone.ClinicalTrials.gov; NCT04375605; Registered 4th May 2020; While overall gastric cancer incidence is decreasing, the incidence of adenocarcinoma of the GEJ has increased in North America and Western European countries , 2. ThisWith disease symptoms appearing late in the course of the disease, most of the patients present with locally advanced or metastatic disease at first diagnosis resulting in dismal 5-year survival rates of 20–30% [Intensive work has been done to increase cure rates in patients with locally advanced GEJ cancers utilising chemotherapy and radiotherapy in the neoadjuvant and adjuvant treatment setting. In patients with resectable tumours, there is evidence supporting the use of both, perioperative chemotherapy and preoperative radiochemotherapy; however, there is an ongoing debate what is the best treatment option and trials thus far have proven inconclusive to favour one approach over the other.According to the most recent German S3-guideline for the treatment of gastroesophageal cancer, both, perioperative chemotherapy and preoperative radiochemotherapy are regarded as reasonable treatment options . In recePerioperative chemotherapy with epirubicin, cisplatin and infusional 5-fluorouracil (ECF)  has beenp = 0.012). 3y OS rate was 48% with ECF/ECX and 57% with FLOT. FLOT also improved PFS . Perioperative complications were 50% with ECF/ECX and 51% with FLOT. 30- and 90-day mortality was 3 and 8% with ECF/ECX and 2 and 5% with FLOT. Regarding toxicities, in the FLOT group, all patients had some type of toxicity (57.1% reporting ≥3 symptoms), however, side effects were generally manageable. The relative effect from FLOT was observed in all subgroups, including elderly patients and signet cell tumours, and was particularly pronounced in AEG type 1 tumours (HR 0.60), Barrett tumours (HR 0.62), T1/2 tumours (HR 0.66) and nodal negative tumours (HR 0.64). FLOT was found to be superior to ECF/ECX in all relevant subgroups and is nowadays widely regarded as the perioperative chemotherapy regimen of choice in esophagogastric adenocarcinoma.In search of an optimized preoperative chemotherapy regimen, the Arbeitsgemeinschaft Internistische Onkologie (AIO) has conducted a phase III trial comparing FLOT with ECF in the neoadjuvant treatment of patients with resectable esophagogastric adenocarcinoma  . FLOT imp = 0.003; HR 0.657) with comparable postoperative morbidity and mortality rates of 46 and 4%, respectively. The benefit of neoadjuvant radiochemotherapy on survival was especially seen for patients with SCC and to a lesser extent for patients with adenocarcinoma. Toxicity of radiochemotherapy was low in terms of hematologic and non-hematologic side effects.Alternatively, the strategy of neoadjuvant radiochemotherapy has been implemented due to the results of the CROSS trial, in which van Hagen and colleagues randomly assigned 368 patients with esophageal cancer (23% SCC and 75% adenocarcinoma) into either a neoadjuvant radiochemotherapy regimen, based on weekly carboplatin and paclitaxel followed by surgery, or surgery alone [With regard to the radiochemotherapy protocol, concurrent radiochemotherapy regimens generally use a combination of cisplatin and 5-fluorouracil or carboplatin and paclitaxel. However, it could be shown that a combination of oxaliplatin and continuous infusion of 5-fluorouracil together with radiotherapy is also well tolerated and efficacious for localized esophageal cancer , 10.With promising results from both, the CROSS and the FLOT4 trial, the question remains whether neoadjuvant chemotherapy or neoadjuvant radiochemotherapy is eliciting the best results in patients with GEJ cancer.A benefit for neoadjuvant radiochemotherapy vs. chemotherapy alone has been suggested from indirect comparisons in meta-analyses but could not be clearly demonstrated in direct comparisons , 12. TwoIn addition, all of the trials have been performed in studies with mixed patient cohorts and before the FLOT regimen and the CROSS protocol as most effective neoadjuvant treatment approaches for GEJ cancers became standard of care.Since both strategies, radiochemotherapy and perioperative chemotherapy provide significant gains in survival, we hypothesize that adding radiochemotherapy to the FLOT regimen will achieve even greater survival gains in the similar patient population.The RACE trial addresses the question if preoperative FLOT induction chemotherapy followed by preoperative radiochemotherapy and postoperative completion FLOT chemotherapy is superior to perioperative FLOT chemotherapy alone in patients with adenocarcinoma of the gastroesophageal junction undergoing adequate oncological surgery.The RACE trial is an investigator initiated multicentre, prospective, randomized, stratified phase III clinical trial and is financially supported by Deutsche Krebshilfe e.V. (German Cancer Aid). Eligible patients will be randomly allocated to one of two treatment groups, i.e. preoperative chemotherapy or preoperative chemotherapy with subsequent preoperative radiochemotherapy, both followed by resection and postoperative completion of chemotherapy Fig. .. The prPatients are deemed eligible for trial participating with histologically confirmed diagnosis of locally advanced, potentially resectable non-metastatic adenocarcinoma of the gastroesophageal junction (AEG type I-III). Patients need to provide written informed consent before trial enrolment and must meet all of the following inclusion and exclusion criteria.Histologically proven, locally advanced and potentially resectable adenocarcinoma of the gastroesophageal junction (GEJ) (AEG I- III): cT3–4, any N, M0 or cT2 N+, M0 (AJCC 8th edition)Patients must be candidates for potential curative resectionECOG performance status 0–1Age ≥ 18 years9/l, platelets ≥100 × 109/l and haemoglobin ≥9.0 mg/dlAdequate hematologic function with absolute neutrophil count (ANC) ≥ 1.5 × 10INR < 1.5 and aPTT< 1.5 x upper limit of normal (ULN)Adequate liver function Adequate renal function (serum creatinine ≤1.5 x ULN)QTc interval (Bazett) ≤ 440 msWritten informed consent obtained before randomizationNegative pregnancy test for women of childbearing potential. Males and females of reproductive potential must agree to practice highly effective contraceptive measures.Evidence of metastatic diseasePast or current history (within the last 5 years) of other malignancies.Evidence of peripheral sensory neuropathy > grade 1 according to CTCAE version 4.03Significant underlying medical conditions that may be aggravated by the study treatment or are not controlledPregnant or lactating femalesPatients medically unfit for chemotherapy and radiotherapyPatients receiving any immunotherapy, cytotoxic chemotherapy or radiotherapy other than defined by the protocolKnown hypersensitivity against 5-FU, leucovorin, oxaliplatin or docetaxelOther known contraindications against 5-FU, leucovorin, oxaliplatin, or docetaxelClinically significant coronary heart disease, cardiomyopathy or congestive heart failure, NYHA III-IVClinically significant valvular defectOther severe internal disease or acute infectionPeripheral polyneuropathy > NCI Grade II according to CTCAE version 4.03Chronic inflammatory bowel diseaseAny of the following renders patients ineligible to participate in this trial:2 d1, leucovorin 200 mg/m2 d1, oxaliplatin 85 mg/m2 d1, docetaxel 50 mg/m2 d1) followed by surgical resection 4–6 weeks after day 1 of the last cycle of neoadjuvant therapy. 6–12 weeks after surgery adjuvant chemotherapy starts with 4 cycles of FLOT .Patients randomized into the perioperative chemotherapy arm receive 4 cycles of neoadjuvant chemotherapy with FLOT every 2 weeks  and continuous infusional 5-FU 225 mg/m2 plus concurrent radiotherapy given in 5/week fractions with 1.8 Gy to a dose of 45 Gy over 5 weeks. Resection is performed 4–6 weeks after last treatment with chemotherapy / radiation. Adjuvant treatment starts 6–12 weeks after surgery and consists of 4 cycles of FLOT .Trial subjects in the investigational arm receive 2 cycles of neoadjuvant induction chemotherapy with FLOT (doses as above) every 2 weeks (4 weeks of therapy) followed by radiochemotherapy beginning at day 21 after day one of the last cycle of chemotherapy. Radiochemotherapy consists of oxaliplatin 45 mg/mFour–six weeks after completion of neoadjuvant treatment, patients in both arms will be scheduled for operation.AEG type I tumours are treated by resection of the proximal stomach and transthoracic esophagectomy with 2-field lymphadenectomy.AEG type II and III tumours are treated by gastrectomy with transhiatal resection of the distal oesophagus and transhiatal lymphadenectomy of the lower mediastinum.For all tumour types, abdominal lymphadenectomy is performed as D2 dissection. The minimum number of lymph nodes to be harvested and pathologically analysed is 16, with 25 lymph nodes recommended.After transthoracic esophagectomy, reconstruction is preferably performed by an intrathoracic anastomosis with a gastric conduit, alternatively by cervical anastomosis or colon interposition. After gastrectomy and transhiatal resection of the distal oesophagus, reconstruction is carried out by esophagojejunostomy.The primary objective is to investigate whether neoadjuvant induction chemotherapy with FLOT followed by radiochemotherapy is superior to perioperative chemotherapy alone in the treatment of resectable GEJ adenocarcinoma in patients undergoing oncologically adequate surgery . The primary endpoint is progression-free survival, recorded as time from randomization until disease-progression, disease recurrence after surgery or death of any cause. Moreover, incomplete (R1 or R2) resection as well as irresectability will be recorded as PFS events. Patients lost to follow up will be censored to the date of last assessment without any such event. Patients will be followed up for a maximum of 60 months after randomization.Overall survival, including survival rates after 1, 3 and 5 years: Overall survival will be measured as time from randomization to death of any cause or last observation.R0 resection rateNumber of harvested lymph nodesSite of tumour relapse Perioperative morbidity and mortality rateSafety and toxicity by NCI CTC criteriaQuality of life (QoL) by using the EORTC QLQ-C30 and the esophagogastric module OG25Screening assessments need to be completed within 4 weeks prior to randomization. Once diagnosis of gastroesophageal adenocarcinoma is confirmed, pre-therapeutic work up includes an electrocardiogram as well as assessment of tumour localisation and size by esophagogastroduodenoscopy and assessment of tumour infiltration and locoregional lymph node involvement based on endoscopic sonography. Computed tomography of the chest, abdomen and pelvis is performed to exclude metastatic disease. For patients with suspected peritoneal tumour seeding, a diagnostic laparoscopy is performed. Within 2 weeks prior to the start of treatment, patients are further evaluated based on their medical history, a physical examination including body weight, height, vital signs and ECOG performance status. Laboratory testing includes a differential blood count and standard clinical chemistry. Patients meeting all inclusion and exclusion criteria can be enrolled and randomized.During neoadjuvant treatment, patient evaluation for toxicity and adverse events is carried out on day 1 of each cycle and includes a physical examination, weight, vital signs, and hematologic and clinical chemistry laboratory tests. Patients receiving radiochemotherapy (Arm B) are assessed on a biweekly schedule with additional assessments on day 1, 15 and 29. Clinical restaging of the disease is performed preoperatively by esophagogastroduodenoscopy with endoscopic ultrasound and computed tomography of thorax, abdomen and pelvis. After completion of surgery, tumours are evaluated for histopathological response  and patiClinical follow up visits will be performed every 3 months for up to 3 years according to the most recent German S3-guideline for the treatment of gastroesophageal cancer . AfterwaFor the chemotherapy group a 3-year PFS of 40% is assumed . An incrProgression-free survival will be compared between both treatment groups with a log-rank test stratified by primary tumour site using a two-sided level of significance of 5%. Patients without an observed event of interest will be treated as censored observations. The primary analysis will be performed following the intention-to-treat (ITT) principle. Kaplan-Meier curves will be shown to illustrate survival functions for both treatment groups. Estimates for median survival and 1-, 3-, and 5-year survival with 95% confidence intervals (CIs) will be presented for both treatment groups. The hazard ratio with 95% CI will be estimated using a Cox regression model under the proportional hazards (PH) assumption with treatment group and tumour site included as fixed effects.Secondary endpoints will be analysed in an explorative manner. R0 resection rate will be compared between both groups using chi-squared tests stratified for tumour site. For continuous outcomes (e.g. QoL scores) a linear regression model including treatment group and tumour site as fixed effects will be fit to the data. Analysis of overall survival will be performed as described for PFS.Toxicities will be evaluated in the respective safety population. Absolute and relative frequencies of adverse events will be reported for each treatment group and for relevant subgroups as different tumour sites and institutions. Confidence intervals for probabilities of adverse events will be estimated. Group comparison will be conducted using Fisher’s exact test.No interim analyses are planned. Efficacy parameters will be analysed at the end of the study unless the DSMB requests an unplanned interim analysis for safety reasons.Incorporation of neoadjuvant treatment strategies in the management of locally advanced gastroesophageal adenocarcinoma has substantially improved clinical outcomes for this patient cohort –7. One oDespite these obvious successes, the overall prognosis remains unsatisfactory with 5-year overall survival rates still less than 50%. Intensive work has been done throughout the world to increase cure rates in patients with locally advanced GEJ cancers utilising chemotherapy and radiotherapy in the neoadjuvant and adjuvant treatment.p = 0.006) and higher complete response rates . However, this came at the cost of a greater level of therapy-related toxicities and did not translate into a significant survival benefit, as demonstrated by a large meta-analysis encompassing five randomized controlled trials with a total of 922 patients [n = 1052) demonstrated a 50% reduction in incomplete resection rates and a borderline significant improvement of the 2-year-failure rate  for those patients receiving neoadjuvant induction therapy before undergoing surgical resection [The currently available evidence has proven inconclusive to favour radiochemotherapy over chemotherapy alone in the neoadjuvant setting –15. Publpatients . In coloesection . TherefoSmall prospective and retrospective trials  are subjThe RACE trial sets out to overcome the limitations previous trials faced focusing on a clearly defined entity, i.e. adenocarcinoma of gastroesophageal junction, using the modern chemotherapeutic regimen FLOT as perioperative treatment and as induction therapy before radiochemotherapy within an adequate sample size to derive meaningful conclusions and ultimately guide clinical treatment strategies.The RACE trial is a multicentre prospective randomized controlled trial investigating the role of preoperative radiochemotherapy added to adequately dosed perioperative FLOT regimen compared to perioperative FLOT chemotherapy alone in patients with locally advanced adenocarcinoma of the gastroesophageal junction undergoing adequate oncological surgery. We hypothesize that addition of neoadjuvant radiochemotherapy to perioperative chemotherapy compared to perioperative chemotherapy alone will result in an increased progression-free survival.


NLRP3 and CARD8 polymorphisms has never been addressed in PCOS yet. We conducted a case-control study conducting of total 169 Slovenian PCOS patients and 83 healthy blood donors. They were genotyped for polymorphisms in antioxidative  and inflammatory pathways genes  using competitive allele-specific polymerase chain reaction (PCR). Logistic regression and the Mann–Whitney test were used in the statistical analysis. SOD2 rs4880, CARD8 rs2043211, and IL1B rs16944 were associated with the risk of developing PCOS. Furthermore, the interactions between CARD8 rs2043211 and IL6 rs1800795 and between IL1B rs1143623 and IL6 rs1800795 also significantly affected the risk for PCOS. With regard to glucose homeostasis, CAT rs1001179, SOD2 rs4880, PON1 rs854560, NLRP3 rs35829419, and TNF rs1800629 were significantly associated with response to the glycemic load. Our data indicate that the genetic variability in the antioxidative and inflammatory pathways influences the development of PCOS and glucose homeostasis in PCOS patients.Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder of multifactorial etiopathology likely to involve the interactions between genetics and lifestyle. Chronic inflammation and oxidative stress (OS) may participate in the pathophysiology of the syndrome. The question of the extent to which OS and inflammation are causally related to the development of the syndrome and metabolic complications remains unanswered. By our knowledge, the role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome as an important trigger of inflammatory pathways and  Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women in the reproductive period as it affects between 4 and 12% of the female population. It has been shown that both genetic and environmental factors play a role in PCOS development and its numerous clinical manifestations  and estimated mass [g], volume [cm3] and surface [cm2] of visceral adipose tissue (VAT). The estimated VAT values were measured using dual-energy X-ray absorptiometry . The PCOM was confirmed if the following ultrasound criteria for PCOM were met: a follicle number per ovary of more than 20 follicles (2–9 mm) and/or an ovarian volume ≥10 mL using transducer frequency ≥8 MHz, or the criterian of ovarian volume ≥10 mL for older ultrasound equipment [Anthropometric data and metabolic parameters were obtained at the beginning of the study before any treatment protocol began. Anthropometric data included height [cm], weight [kg], waist circumference [cm], BMI [kg/mquipment . A standSOD2 rs4880, CAT rs1001179, PON1 rs854560 and rs662) and six related to inflammation . The genotype distribution for all investigated polymorphisms was in Hardy-Weinberg equilibrium (HWE) . PON1 rs854560 and rs662 were not in linkage disequilibrium (LD) (R2 = 0.202). IL1B rs1143623 and rs16944 were slightly linked, but below the cutoff of 0.8 (R2 = 0.733).In molecular genetic analysis, genomic DNA was isolated from peripheral blood leukocytes using the FlexiGene DNA kit . Selected single nucleotide polymorphisms (SNPs) were genotyped using a fluorescent-based competitive allele-specific polymerase chain reaction (KASPar) assay  or real-time PCR-based Taqman SNP genotyping assay  following the manufacturer’s instructions. In total, we genotyped four polymorphisms related to OS  values. In the analysis of polymorphisms influence on anthropometric characteristics and glucose homeostasis, only the dominant model was used. The differences in the distribution of categorical data and possible interactions between genotypes were determined by using logistic regression. When analyzing continuous variables, the Mann–Whitney test was used. The differences were considered statistically significant when the p-value was below 0.05. All statistical analyses were performed using IBM SPSS Statistics version 21.0 .In the statistical analysis, the distribution of categorical data was presented with the frequencies, while the continuous variables were presented with median and interquartile range (25–75th percentile). A Chi square test was used to assess deviation from HWE. In the analysis of PCOS risk, the wild-type genotype served as a reference, and its frequency was then compared with the frequencies of other genotypes in additive and dominant models. In In conclusion, the importance of OS and inflammation and their causal relationship in PCOS development have been speculated for a long time, but so far, most studies have only measured different markers of OS and inflammation in PCOS patients. Our data indicate that genetic variability in genes coding for antioxidant enzymes and inflammatory pathway mediators not only modifies the risk for PCOS but also influences the metabolic characteristics of PCOS patients. More extensive studies with age and BMI-matched control and study groups could explore the role of the selected polymorphisms in PCOS pathogenesis even further. Furthermore, better understanding of the role of genetic variability in these pathways may be of importance for everyday clinical practice through identifying patients’ groups that are at risk for PCOS development and predicting their clinical progression and their risk for metabolic complications.


Changes of the ST segment are commonly used as predictors of the culprit vessel during an acute myocardial infarction. In case of combined ST elevation in both inferior and anterior leads, these changes can be due to a distal occlusion of a “wrapped” left anterior descending artery (LAD) or a two-vessel disease. Our case of anterior wall myocardial infarction with inferior ST elevation and anterior ST depression shows that electrocardiographic changes during acute myocardial infarction cannot always be explained by logical sequelae of the injury current, vessel anatomy, and their irrigation territory. ST segment changes, whether elevation or depression, as well as their distribution are commonly used as predictors of the culprit vessel during an acute myocardial infarction. ST elevation in inferior leads suggests an acute occlusion of the right coronary artery (RCA) or the left circumflex coronary artery (LCx). In case of combined ST elevation in both inferior and anterior leads, these changes can be due to a distal occlusion of a “wrapped” left anterior descending artery (LAD) or a two-vessel disease . There aA 30-year-old male patient with no previous history of cardiovascular disease, cardiovascular risk factors, or family history of cardiovascular disease presented in emergency department with retrosternal chest pain not related to exertion that had started four hours before. There was no pain radiation, nausea, or vomiting. He had no recent history of infectious disease (as a pericarditis/myocarditis trigger). On physical examination, there were no abnormal findings. The initial electrocardiogram (ECG) showed 2 mm ST segment elevation in leads II, III, and aVF; 1 mm in V6; and horizontal ST depression in aVL and V1-3 . His bloDuring acute anterior wall myocardial infarction, elevation of the ST segment is usually seen in the anterior leads. ST segment depression is sometimes observed simultaneously in the inferior leads which is generally considered to be a reciprocal change of ST elevation in aVL related to anterolateral extension of the ischaemia of D1 branch irrigation territory .Inferior ST elevation during acute anterior myocardial infarction is unusual but explainable when the “wrapped” LAD is occluded distal to its first diagonal branch, appearing with a combination of a transmural ischaemic myocardium in the anterobasal and anterolateral wall of a lesser degree together with transmural ischaemic myocardium in the inferior wall .Sapin et al. explained the ST segment changes in “anterior-inferior” myocardial infarction with the distal occlusion in the LAD which allows the more proximal patent branch vessels to perfuse the anterior wall resulting in a smaller mass of ischemic anterior wall myocardium, thus smaller anterior wall injury current and less reciprocal inferior ST segment depression. Inferior wall injury current, as a result of occluded “wrapped” LAD, must be combined with the weak reciprocal of a smaller anterior injury current to generate the ST elevation in the inferior leads . Sasaki Our case describes a rare form of anterior myocardial infarction with inferior ST elevation with no deviation in coronary anatomy and right-dominant system. Also, there was no inferior wall myocardial infarction and no ST elevation in anterior leads. The patient is a PAI-1 4G homozygote, which some authors consider as an independent risk factor for acute myocardial infarction in young patients . To our Inferior ST elevation in an acute myocardial infarction of anterior wall is usually combined with anterior ST elevation. Both of the electrocardiographic changes are caused by occlusion of a “wrapped” LAD distal to D1 branch. Our case of anterior wall myocardial infarction with inferior ST elevation and anterior ST depression shows that electrocardiographic changes during acute myocardial infarction cannot always be explained by logical sequelae of the injury current, vessel anatomy, and their irrigation territory.


UBC and PFDN2, which may be key players in the regulation of differential gene expression in treatment responders and non-responders. Taken together, identifying the gene expression signature may advance our understanding of the molecular mechanisms of azacitidine and may also serve to predict patient responses to drug treatment.Allogeneic stem cell transplantation is currently the only curative treatment option for myelodysplastic syndromes (MDS). Pre-transplant debulking treatment have been employed for advanced MDS and we previously reported that marrow response (blast ≤ 5%) following the bridging therapy with hypomethylating agent was an independent favorable factor for survival; however, it is still not clear which patients will respond to hypomethylating agent and which genomic features can predict the response. In this study, we performed RNAseq for 23 MDS patients among which 14 (61%) and 9 (39%) patients showed marrow complete remission and primary resistance to azacitidine, respectively. Differential expression-based analyses of treatment-naive, baseline gene expression profiles revealed that molecular functions representing mitochondria and apoptosis were up-regulated in responders. In contrast, we identified genes involved in the Wnt pathway were relatively up-regulated in non-responders. In independent validation cohorts of MDS patients, the expression of gene sets specific to non-responders and responders distinguished the patients with favorable prognosis and those responded to azacitidine highlighting the prognostic and predictive implication. In addition, a systems biology approach identified genes involved in ubiquitination, such as  The ineffective hematopoiesis is associated with elevated apoptosis of hematopoietic clones, a phenomenon that may be related to signaling mediated by TRAIL ligands/receptors2, FAS molecules3 and/or myelosuppressive TGFβ4. Excessive apoptosis occurs in the early stages of MDS and declines as MDS progresses toward the leukemic stage. About one third of MDS patients progress to AML, which is probably associated with expansion and evolution of subclones with certain mutations at the stem cell level6. The genetic profiles of individual patients are heterogeneous and an individual patient’s unique genetic makeup may impact their clinical phenotype, prognosis, and response to therapy. An MDS patient’s genome frequently harbors somatic mutations which may contribute to disease progression; however, there is no single gene that appears to be sufficient to elicit disease and the majority of genes mutated in MDS are present in fewer than 5% of cases, highlighting the complexity of the disease8. These heterogenity of MDS have led to the development of risk-based stratification systems, and treatment options such as hypomethylating agent (HMA) or allogeneic stem cell transplantation (SCT) are selected systematically according to risk groups11.Myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by peripheral blood cytopenia due to ineffective hematopoiesis and by their potential to evolve into acute myeloid leukemia (AML)13, but many issues, including determining the optimum dose or duration of treatment, remain to be addressed. Importantly, it is still not clear which patients will respond to HMA or not. A number of predictive genomic markers of the response to HMA treatment have been reported among the frequent genomic aberrations in MDS genomes. For example, mutations in genes involved in DNA methylation  and genes encoding epigenetic regulations  have been proposed to predict AZA sensitivity16, but the clinical relevance of these associations remains controversial19. A DNA methylation-based scoring system using a suite of 10 genes has been proposed, but while the score was correlated with patient survival, it could not predict clinical responses to demethylating agents20. The expression of a number of other genes, such as BCL2L1021, FAS22, and PI-PLCbeta123, may also be indicative of HMA activity. Moreover, associations between repressed expression of anti- DNMT1 miRNAs and HMA resistance have been noted24. All of these findings have contributed substantially to a better understanding of individual variability in HMA responses among MDS patients; However, mutation, methylation, or expression information involving limited genes are not full enough to understand disease heterogeneity with respect to different response to HMA treatment. In this regard, genome-wide profiling of mRNA expression may facilitate comprehensive prediction of drug sensitivity.The use of HMA, such as azacitidine (AZA) and decitabine, has become a standard treatment for higher-risk MDS patients, as HMA confer a survival benefit over conventional care25. Several retrospective studies have shown that pre-SCT HMA could be a feasible alternative to induction chemotherapy28 even for patients with excessive blasts or AML29. As the influence of HMA response on transplant outcomes is still controversial, we previously completed a retrospective analysis of 98 patients who received HMA for higher-risk MDS with > 5% marrow blasts30. Our study showed that continued marrow response at the time of SCT was an independent positive predictor of overall and disease-free survival after transplantation. In light of our findings and those of another study in which a positive correlation between marrow clearance and genomic mutations was reported31, we aimed to identify the molecular markers that could predict a patient’s marrow response to HMA.One important usage of HMA is to serve as a bridge to SCT by diminishing marrow blasts to an acceptable level prior to transplantationIn this study, we performed RNAseq-based gene expression profiling of advanced MDS patients with excess blasts (EB) prior to their receiving AZA treatment. Comparison of gene expression profiles between 14 responders and 9 non-responders revealed a number of molecular functions  were relatively activated in responders. Functional scores of these molecular pathways were also correlated with patient survival in an independent MDS cohort, and the results suggested their potential prognostic value.n = 9) and MDS-EB-2 (n = 14) before SCT were enrolled in the study (Table n = 6) or MDS-EB-2 (n = 2) and 1 case with stable disease without any hematological improvement (SD-HI) after 5 cycles of AZA treatment. The responder group consisted of cases with complete remission (CR) (n = 7) or marrow CR (mCR) with or without any hematological improvement (n = 7). The patients’ bone marrow derived mononuclear cells were subjected to transcriptome sequencing by RNAseq. The sequencing-related information is presented in Supplementary Table A total of 23 patients who received AZA for MDS with excess blasts 1 . The hierarchical clustering of 300 DEGs was able to segregate responders and non-responders, with the exception of one outlier . To summarize the identified GO categories by taking into account the redundancy in GO categories, we measured the extent of overlap for all possible pairs of the 22 GO categories identified above. Of note, all possible pairs of the 22 GO categories showed significant overlap between DEGs , which suggested the gene–gene co-expression networks were highly interconnected with respect to GO functional categories. The 22 GO categories are illustrated in a network diagram  with overlapping DEGs for NDUFS7, COX8A, UBA52, UQCR11, NDUFA2, NDUFB7, NDUFB8, NDUFA7, NDUFB2, COX4I1, NDUFA13, ETFB, NDUFA1, NDUFB9, NDUFA11, and UBC. Oxidative stress and mitochondrial dysfunction have been postulated to play a role in the development of MDS32. Although the association between sensitivity to AZA and mitochondrial dysfunction has not been clearly defined, a number of reports suggest that increased oxidative phosphorylation relative to glycolysis may indicate metabolic plasticity of tumor cells and is associated with the resistance to chemotherapy in colorectal cancers33 and targeted therapeutics in melanomas34.We first selected differentially expressed genes (DEGs) between responders and non-responders  to identify molecular functions for which the gene members are differentially expressed between AZA responders and non-responders. Table 8. Thus, it is reasonable to assume that elevated expression of apoptosis-related genes at baseline may predict a favorable prognosis and responsiveness to AZA. In our study, we found no significant differences in FAS expression between responders and non-responders , or between BCL2L10 and PLCB1 expression , both of which have previously been proposed as expression markers of AZA sensitivity23. In our study, only two genes, BAX and IFNG, had significantly different expressions between the responders and non-responders . Wnt/β-catenin signaling has been presumed to be involved in the ineffective hematopoiesis of patients with myeloid neoplasms with 5q deletions, and has also been presumed to have therapeutic implications in MDS through inhibition of this signaling35. Our results further suggest that the mRNA expression levels in Wnt/β-catenin pathway genes including a number of frizzled receptors may indicate the responsiveness to AZA with similar implications in MDS-derived stromal cells35. It is postulated that up-regulated expression of Wnt pathway genes at baseline in non-responders encodes cellular resistance to AZA treatment. Other GO categories that showed substantial differences in expression with respect to HMA responses are shown in Supplementary Fig. Two examples of GO categories  are selected to show their enrichment plots as well as their expression heat maps and leading-edge genes Fig. b,c, resp36 to identify the genes related to the mechanism of action (MoA) of AZA. In this process, the level of perturbation was measured for individual genes and their interacting partners in regulatory networks by comparing the gene expression profiles of responders and non-responders following drug perturbation. The 20 most significant candidate MoA genes are listed in Table UBC and PFDN2 as candidate MoA genes whose protein interactions with interacting partners were the most perturbed in the context of the regulatory network.In addition to assessing DEG and GSEA, we further employed a DeMAND network-based system biological approach37 and 32 patients with AZA treatment history 39. For survival (GSE58831), MDS patients whose gene expression profiles resembled those of AZA responders in our study (‘responder-like’) showed better survival . This result suggests that the baseline expression profile from AZA responders may be associated with a favorable prognosis in MDS patients highlighting the prognostic implication of the identified genes in our study. For AZA responsiveness (GSE77750), the clustering using identified markers segregated the 32 MDS patients into ‘responder-like’ and ‘non-responder-like’ cases with the modest level of prediction accuracy 0.687  to test the prognostic values of the markers. Although not significant, the expression of MoA genes was able to segregate the MDS patients with a substantial difference in overall survival suggesting that the MoA genes, in spite of their small size, may be utilized for prognostic markers , although recent study by Shiozawa et al. showed the concordance of expression levels for selected genes between bone marrow CD34 + cells and mononuclear cells from MDS patients44. In addition, advanced MDS patients with excess blasts who were treated with HMA were only included in our study, while all subtypes of MDS patients with no information on treatment (GSE58831) or AZA treated AML patients, not only MDS patients (GSE77750) were analyzed in validation cohorts. Actually, when we selected EB patients only in validation cohort (GSE58831), the prognostic impact according to the expression of marker genes was not significant  and as protein chaperone (PFDN2). It has been previously reported that ubiquitination is implicated in histone modification45 and the ubiquitin–proteasome pathway is associated with the stability of the DNMT1 protein, findings which indicate a potential molecular link between ubiquitination and the demethylating agent AZA46. Therefore, we may assume that UBC may have a role in the regulation of gene expression associated with AZA treatment.The DeMAND algorithm was employed as a systems biology approach to identify network-level perturbation and to reveal key MoA genes. Through this process, Taken together, the markers for the AZA sensitivity identified in this study may predict drug responsiveness prior to AZA treatment and may have general prognostic implications.2/day for 7 consecutive days) for MDS with > 5% marrow blasts and cases showing extremes of treatment response (marrow complete remission versus primary treatment failure) were screened. Response to treatment was assessed using the modified International Working Group response criteria47. Cases achieving CR or mCR with or without hematological improvement were categorized as responders, while non-responders consisted of those who experienced primary treatment failure defined either by primary disease progression or stable disease without hematological improvement (SD-HI)48. Minimum 4 cycles of AZA was administered before response assessment of SD-HI, whereas assessment of CR, mCR or disease progression was allowed to assess even before the 4th cycles. The patients did not receive previous treatment before AZA for their MDS with excess blasts. Patients with bone marrow samples available for research purposes were enrolled in the final study population. This study was approved by the Institutional Review Board of the Seoul St. Mary’s Hematology Hospital at the Catholic University of Korea, and complied with the tenets of the Declaration of Helsinki. Informed consent was obtained from all subjects.To identify molecular markers in MDS genomes associated with marrow responses following pre-transplant bridging treatment with AZA patients who received the standard schedule of AZA . The expression of each gene was represented in terms of fragments per kilobase million (FPKM) and used for the subsequent analyses. RNA sequencing data have been submitted to the Sequence Read Archive (SRA) under accession number of PRJNA650236.Bone marrow samples 10 mL) were mixed with 0.3 mL of heparin to prevent coagulation, then diluted with 20 mL of phosphate‐buffered saline . The cells were then fractionated on a Lymphoprep density gradient  through centrifugation at 600 g for 10 min. Interface mononuclear cells were isolated and washed with PBS. An erythrocyte (RBC) lysis buffer  was then added to destroy contaminating RBCs. The RNA was isolated using the standard Tri-Reagent1 protocol. Preparation of mRNA libraries was performed using an illumina (REF. RS-122-2101 ~ 2) TruSeq stranded kit as manufacturer’s recommendation. The sequencing was performed using the NextSeq500 platform . Paired-end 75 bp sequencing reads were generated and the sequencing information is presented in Supplementary Table 0 mL were51  was used to identify differential expression of genes associated with specific molecular functions between drug responders and non-responders52. For molecular functions, we used Gene Ontology gene sets as available in MSigDB .Gene set enrichment analysisSupplementary Information.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Table 1.Supplementary Table 2.


There have been a number of studies showing that myocardial sodium elevation subtly affects mitochondrial function. During contraction, mitochondrial calcium (Camito) stimulates a variety of TCA cycle enzymes, thereby providing reducing equivalents to maintain ATP supply. Nai elevation has been shown to impact Camito; however, whether metabolic remodelling in HF is caused by increased Nai has only been recently demonstrated. This novel insight may help to elucidate the contribution of metabolic remodelling in the pathophysiology of HF, the lack of efficacy of current HF therapies and a rationale for the development of future metabolism-targeting treatments. Here we review the relationship between Na pump inhibition, elevated Nai, and altered metabolic profile in the context of HF and their link to metabolic (in)flexibility and mitochondrial reprogramming.Elevated intracellular Na (Na With few effective treatments available, heart failure (HF) affects over 64 million people worldwide carrying the annual death toll of 17.5 million lives –6. Thus,E-C) coupling including Nai elevation are characteristic features of pathological cardiac remodelling and underpin contractile dysfunction in HF.Numerous molecular mechanisms have been proposed that could contribute to the development of HF and these include an energy deficit following metabolic reprogramming. In a series of precisely regulated enzymatic reactions, heart muscle highly efficiently converts chemical into mechanical energy . This fa2i is reported to be significantly higher at 10-20 mM  (Table may vary . This elThe low intracellular Na is maintained by the activity of the Na/K ATPase or Na/K pump (NKA). The energy invested by the cell in driving the Na/K ATPase, and maintaining this high transmembrane electrochemical gradient, creates a ‘battery’ that the cell then exploits to drive ions, substrates and amino acids into (symports or co-transporters) or out of (antiports or exchangers) the cell . Each ofα and β subunits found in the peripheral sarcolemma as well as T-tubules. α subunit is a highly conserved 110 kDa non-glycosylated protein. On the inner membrane it contains the ATPase domain and on the outer membrane ion-binding and ouabain-binding domains. β subunit is a ~55 kDa glycoprotein ..2 supply4i . In. Inm stii elevation significantly reduces both diastolic and systolic Cam. Consistently, the percentage of NAD(H) in the control group was maintained at ~62%, but in the high Nai group was significantly reduced. Despite changes in Ca and [NADH], Nai elevation did not change the mitochondrial membrane potential (Δψm). Another premise of their work was that failure to maintain pyridine nucleotide reduction (NADPH) and in turn antioxidant flux due to high Nai contributes to reactive oxygen species (ROS) overflow which is readily corrected by lowering Nai in failing heart cells [i plays an important role in regulation of cardiac energy homeostasis. However, which metabolic pathways are most affected by Nai overload and whether any of these changes were truly reflective of the regulatory mechanism in the beating heart remained to be shown.In isolated guinea pig cardiomyocytes Maack et al.  have short cells . Collectmito increases Namito and leads to release of Cam from calcium phosphate precipitates as well as Na-dependant reduction of the fluidity in the inner mitochondrial membrane. Consequently, alterations in the membrane fluidity caused a reduction in ubiquinone mobility between ETC II and III leads to ROS production. Inhibition of Na/Camito was shown to block all these events.A recent study by Hernansanz-Agustin et al.  has offe6mito) activates key Ca-sensitive TCA cycle regulatory enzymes (CaDHmito) including pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase and the NAD-linked isocitrate dehydrogenase  as well as redox signalling regulator  or aetiology  and deterioration in function. This was observed in models of chronic and acute Na elevation: PLM3SA mouse with NKA inhibition, left ventricular hypertrophy (TAC model) and pharmacological NKA inhibition with ouabain  but may also improve the ATP supply.However, given that we have observed depletion of the metabolic substrates including lactate as well as depletion of amino acids, it is possible that the metabolic adaptation observed in the long run may not be sufficient to maintain PCr/ATP ratio comparable to the healthy hearts. Mathematical simulations support this hypothesis based on flux analysis. However, additional tracer experiments are needed to assess flux distributions under these conditions. In silico work showed that the impact of elevated Na process . However process . Our resIn our perfused murine preparations glucose was the preferred substrate under physiological conditions. Whilst surprising, it is consistently observed that murine hearts perfused in isolation have a baseline preference for carbohydrates, potentially due to differences in oxygen needed to fully oxidize carbohydrates vs fatty acids  –82. Furt7i has inadvertently been a HF therapeutic target for at least 200 years. Glycosides are not only present in plants such as the foxglove , but are also endogenously found in animals under physiological conditions  and are elevated in chronic kidney disease patients [i and the resetting of Na/Ca exchange leading to cellular Ca loading. While historically inotropic agents were considered to offer symptomatic relief to HF patients, it is relatively recently that this was recognised as ‘flogging a dead horse’ [Since William Withering’s recognition of the efficacy of cardiac glycosides, Napatients  and HF [patients . The pospatients , the eled horse’ . Recent d horse’ . Lowerind horse’ ,89. The l-carnitine) could also further help to elucidate the role these metabolic alterations play in transition from compensated hypertrophy to HF [While therapeutically reducing elevated Na may have multiple benefits, it may also be possible to target the consequences of Na elevation more directly and specifically to target deranged metabolism. In spite of the substantial data to support the pharmacological targeting of the substrate switch, successful translation to the clinic has been limited, with the exception, perhaps, of the cluster of drugs which target post-ischaemic myocardial energetic efficiency . These ihy to HF .i overload by pharmacological activation of NKA may be a more superior therapeutic strategy for attenuation of metabolic remodelling in HF with potentially fewer off-target effects [i would be expected to exacerbate pre-existent metabolic remodelling. Inhibitors of renal sodium-glucose co-transporter (SGLT2i) have been used at the successful type 2 diabetes treatment with cardioprotective, glycaemic control-independent effects observed in HF patients. It was widely postulated that SGLT2i produce their positive effects in HF by reducing Nai by direct inhibition of cardiac NHE1 activity [31P and 23Na NMRS of the mouse, rat and guinea pig hearts) to assess the impact of SGLT2i empagliflozin on intracellular Na and NHE1. Our study has shown that SGLT2 inhibitors have no effect on Nai concentration nor NHE1. This has been confirmed for the whole range of therapeutic empagliflozin doses and other SGLT2i’s. Therefore, any beneficial effects of SGLT2inhibitor family in failing hearts cannot be attributed to their action on myocardial NHE1 or intracellular Na [Given that mitochondrial Na/Ca exchange inhibitor CGP37157 would treat a consequence of sodium elevation rather than a primary cause, our recently published work suggests that reducing Na effects ,102. Ouractivity ,104. In lular Na .i elevation could either preclude the origin or change the course of metabolic remodelling in HF. This hypothesis warrants precision medicine approach and extensive further work including development of pharmacological agents that target these interconnected pathophysiological events.Given the complexity of heart metabolism, it remains to be seen whether early prevention of myocardial Na


Improvement in traits of agronomic importance is the top breeding priority of crop improvement programs. Majority of these agronomic traits show complex quantitative inheritance. Identification of quantitative trait loci (QTLs) followed by fine mapping QTLs and cloning of candidate genes/QTLs is central to trait analysis. Advances in genomic technologies revolutionized our understanding of genetics of complex traits, and genomic regions associated with traits were employed in marker-assisted breeding or cloning of QTLs/genes. Next-generation sequencing (NGS) technologies have enabled genome-wide methodologies for the development of ultra-high-density genetic linkage maps in different crops, thus allowing placement of candidate loci within few kbs in genomes. In this review, we compare the marker systems used for fine mapping and QTL cloning in the pre- and post-NGS era. We then discuss how different NGS platforms in combination with advanced experimental designs have improved trait analysis and fine mapping. We opine that efficient genotyping/sequencing assays may circumvent the need for cumbersome procedures that were earlier used for fine mapping. A deeper understanding of the trait architectures of agricultural significance will be crucial to accelerate crop improvement. Genetic improvement in crop plants is a continuous process of developing improved cultivars to meet the ever-increasing human demand for food, nutrition and energy. Natural variations available for important agronomic traits were utilized in crop improvement activities across the world over the century. Mendel’s experiments on genetic inheritance in the nineteenth century constituted the scientific basis for understanding genetics of plant traits and crop improvement through systematic plant breeding. Majority of the agriculturally important traits are complex or quantitative in nature . With the latest SNP genotyping platforms in place, it is now possible to genotype tens of thousands of samples in a short span of time. In this review, we discuss and critically appraise the efforts to fine-map QTLs, cloning QTLs/genes and identification of candidate/causative genes in the pre- and post-NGS era.Three factors, viz. population size  is the first model crop plant species where the use of RFLP markers and QTL identification were reported , amplified fragment length polymorphism (AFLP), SSR markers were used for trait dissection. Tomato  clone on the physical map. The clones thus identified were used for sequencing and developing DNA markers within the QTL region Fig. . A limitIncreasing adoption of NGS-based assays for population genotyping has facilitated high-density linkage mapping in various crop species  in the pre-NGS era demanded considerable time and the effort of few technicians in comparison with the current NGS assays that allow highly saturated genetic maps  to be constructed within few months with modest technical efforts  for gene discovery Whole transcriptome sequencing of contrasting bulks is referred to as BSR-Seq. The technique is particularly important for crops with large and complex genomes like wheat where resequencing still remains cost-ineffective  gene of maize to ~ 2 Mb region and a single gene myb transcription factor was reported in this region. This gene was reported to control the genes involved in long-chain fatty acid synthesis in maize  with sequencing methods. This approach has been established as highly efficient for rapid discovery of candidate genes for the trait of interest. By allowing placement of QTL within a smaller genomic segment, it facilitates both detection of QTL and its fine mapping at a stretch. The procedure involves creation of extreme bulks by selecting lines with extreme low and high values of trait from a segregating population. The pools along with one of the parental genotypes are subsequently sequenced using NGS. A reference genome assembly for the parental genotype is developed based on sequencing data. Subsequently, SNP index for each bulk is computed based on the number of reads for a SNP that differs from the SNP in the parent reference assembly.  Similarly, if all reads have a different nucleotide compared to the reference parent nucleotide, then the SNP index is 1 (10/10). Based on these analyses, Δ-SNP index value of high and low bulks is calculated and the candidate genomic region for the trait of interest is identified. This approach was successfully demonstrated for faster identification of QTLs for blast resistance in rice (Oryza sativa)  , Singh et al. (Cajanus cajan), sequencing of resistant and susceptible bulks in combination with WGRS data of four additional genotypes elucidated candidate genes associated with Fusarium wilt and sterility mosaic disease resistance  identification of the sequences unmapped with the reference genome, (ii) delineation of the candidate genomic segment with MutMap approach, (iii) creation of a de novo assembly of the target region through combining short reads pinpointed in steps i and ii, (iv) alignment of the bulk-sequenced reads  to the ‘P + scaffolds’ reference  identification of the causal mutation within the gap region , which involves mutagenesis and screening for mutants followed by chromosome sorting of the selected mutant and sequencing the specific chromosome to identify the causative mutation  technology for rapid cloning: RenSeq is a NBS-LRR gene-specific R gene enrichment method, which allows rapid identification of disease resistance genes by targeted resequencing. This technique includes bait design using known NBS-LRR gene families followed by sequencing of the enriched samples NBS-LRR and genome annotation. As a proof-of-concept study, Jupe et al. (Solanum tuberosum) and tomato. In this study, target enrichment library was prepared using 523 NB-LRR-like sequences from potato genome, 57 tomato NB-NRC domains, 9 characterized NB-LRR types from tomato, tobacco (Nicotiana tabacum) and pepper (Capsicum annum). The enriched samples against genomic DNA of the sequenced S. tuberosum Group Phureja clone were sequenced, and annotation was carried out. This resulted in successful enrichment of NB-LRRs from 438 to 755. (ii) MutRenSeq: Subsequently, Steuernagel et al.  that combines chemical mutagenesis with exome capture and sequencing for rapid cloning of resistance genes such as stem rust resistance genes Sr22 and Sr45 in hexaploid wheat. MutRenSeq significantly reduces the duration of gene cloning process from 5–10 years to 2 years. (iii) SMART-RenSeq: Witek et al. (Phytophthora infestans (Rpi-amr3i) causing late blight disease in potato. (iv) AgRenSeq: In order to develop broad range of disease resistance in domesticated crops, R genes from wild relatives can be utilized. To achieve this, association analysis was combined with RenSeq approach to develop AgRenSeq method  via long-range assembly follows an approach similar to MutChromSeq where prior information about the mapped gene (flanking markers of a QTL) and its chromosomal location is used for chromosome sorting and sequencing. Thind et al.  for sgRNA designing. The mutant lines (sapk2) were more sensitive to drought and reactive oxygen species (ROS) than wild-type plants. This study suggested that the OsSAPK2 will be a candidate gene for drought tolerance in rice. Recent reviews highlight increasing contributions of CRISPR/cas9 toward novel gene identification and characterization in various crops  and double haploid (DH) have been used for QTL identification .Another multi-parent mating design NAM involves crossing one common reference genotype with diverse founders to generate a series of “interconnected” segregating inbred families. Like MAGIC, NAM offers the advantages of both linkage analysis and association mapping approaches while overcoming the shortcomings of both approaches. The utility of NAM design for QTL mapping is well established in maize  is the first plant genome sequenced, and the availability of the genome sequence of Arabidopsis has brought tremendous changes to the methods of fine mapping and cloning . Recombinants were identified using two flanking SSR markers. The marker numbers were increased by four yeast artificial chromosome (YAC) clones from this region. The cleaved amplified polymorphic sequence (CAPS) markers were generated, and further progeny testing delimited the FLC region to a 620 kb interval. Further, analysis of BAC clones in this region delineated a 10–20 kb region with three genes, among which MADS box transcription factor was found to play an important role in flowering in Arabidopsis. This research took almost 5 years from coarse mapping to positional cloning of the flowering gene. The availability of genome sequence of Arabidopsis provided ample marker resources for fine mapping and QTL cloning for the trait of interest , which identified a candidate gene ORFX, and the underlying mechanism was elucidated with complementation test of this gene. In another study, 7000 F2 lines were used for fine mapping of sugar content in tomato  was also cloned  . Using map-based cloning approach, Dai et al.  into O. sativa ssp indica cv GuangLuAi4 (GLA4) demonstrated to have a large impact on reducing awn length and increasing tiller and grain numbers in domesticated rice  resistance QTL to short interval of 1.2 cM was reported in wheat . This study also highlighted micro-collinearity among wheat, rice and barley with respect to the genomic region controlling FHB resistance. Similarly, positional cloning of VRN1 gene for vernalization in wheat employed a large population for progeny testing (3095 F2 plants) and comparative physical maps of rice and sorghum for collinearity analysis of VRN1 region  gene which explains the evolutionary changes during maize domestication. This gene was found to play an important role in repressing the growth of axillary organ and enable the formation of female inflorescences. Initially, QTL for tb1 was mapped on F2 population; further, the QTL was introgressed into different genetic backgrounds to validate the QTL and complementation test has confirmed the role of tb1 in maize architecture. A similar analysis of fruit case/ear structure on 3106 F2 plants delimited a single gene, teosinte glume architecture (tga1) into 1024 bp controlling ear structure from a wild teosinte to domesticated maize  in maize. The cloned region was confined to ~ 2 kb noncoding region positioned 70 kb upstream from Ap2-like transcription factor. A large F2 population comprising 10,000 lines derived from N28 × NIL C22-4 was used for the QTL cloning study. NIL C22-4 was obtained through the twenty cycles of straight backcrossing of N28 and Gaspé Flint (an early flowering variety). Tassel and ear architecture were dissected using an early-generation population genotyped with low-coverage GBS assay  was identified to be the candidate gene of Nss associated with nicosulfuron sensitivity in maize  for powdery mildew resistance using RFLP markers on backcross lines. High-resolution mapping of Rym4/Rym5 locus conferring resistance to the barley yellow mosaic virus complex  were reported by Pellio et al.  gene was cloned using DH lines and a large mapping population comprising 6720 lines  in barley , was provided through mutant screens, gene expression and gene silencing experiments. More recent use of WGRS data of 106 soybean accessions by the same group has established major role of CNVs in rhg1 (GmSNAP18) and Rhg4 (GmSHMT08) loci in combination with epistasis and promoter variation for broad-based resistance against SCN  and gene overexpression analyses. A similar QTL-Seq approach in groundnut was associated with 2.4 Mb and 0.74 Mb genomic regions on the pseudomolecules B05 and A09, respectively, with fresh seed dormancy trait  can easily identify the well-annotated genes in those regions. In fact, rapid trait mapping approaches such as QTL-Seq can identify the well-annotated genes directly on genome assembly. Such methodological leaps in our opinion indicate non-requirement of traditional cloning methods that seek initial coarse mapping of the genomic region followed by fine mapping to reach candidate loci. The recent post-NGS technologies definitely can help to reduce the time of QTL cloning, and more importantly, one can even bypass the standard fine mapping processes as the primary mapping itself could pinpoint the genes in the QTL region with great precision.https://www.lifetechnologies.com/in/en/home/life-science/agricultural-biotechnology/discovery-of-high-density-molecular-markers.html). The resources and time invested to attain candidate genes have been reduced tremendously with these NGS technologies. In the recent past, fine mapping of QTL has become less complicated and rapid due to a variety of reasons: (i) availability of reference genomes for majority of crop species, (ii) availability of high-resolution mapping populations such as MAGIC and NAM, (ii) possibility of construction of genetic maps and undertaking QTL analysis with high marker densities, (iii) possibility of landing directly to genes in QTL regions using rapid trait mapping approaches such as QTL-seq and RenSeq, (iv) availability of gene annotation information for majority of crops to easily shortlist possible genes from the candidate gene lists and (v) availability of mutant populations in several crops and possibility of using fast gene editing approaches for validating gene function. The projects that had taken considerable time (10–15 years) to fine-map, clone QTL and identify a candidate gene now can be completed within a time of 10–20 months.Fine mapping and QTL cloning were instrumental in understanding the functional mechanism of important plant phenotypes in the past decade. However, sequencing technologies have revolutionized genomics and breeding research in the last decade than in the last 150 years (