Acute graft-versus-host disease (aGVHD) is a serious complication after stem cell transplantation and is associated with high non-relapse mortality. If steroid treatment as first-line therapeutic approach fails, treatment options are limited. In retrospective studies, ruxolitinib, a selective Janus kinase 1/2 inhibitor as well as extracorporeal photopheresis (ECP) could show high efficacy in treatment of steroid refractory acute and chronic GVHD. Here, we report single-center experience of combining JAK-inhibitor treatment with ECP in 18 patients with severe steroid refractory aGVHD of lower GI-tract. The treatment was well tolerated and no severe cytopenia (grade IV) occurred, in three patients grade III cytopenia could be observed. Response was complete or partial in 44% and 11%, respectively, resulting in an estimated 2 year overall survival of 56%. Steroids were tapered rapidly with a median time of 2 days for halving of dosage avoiding additional steroid-associated side effects. Under treatment with ruxolitinib and ECP, an increased level of regulatory T cells could be observed elucidating direct effects of this treatment on immune response. Acute graft-versus-host disease (aGVHD) is a frequent complication and the major cause of non-relapse morbidity and mortality after allogeneic stem cell transplantation (ASCT). Even though the survival rates after ASCT have significantly increased over the past decades, the rate of developing aGVHD remains high with an occurrence in ~50% of all ASCT cases . Main unRuxolitinib is a selective, small molecule Janus kinase (JAK) 1/2 inhibitor approved for therapy of advanced myelofibrosis and for polycythemia vera patients with inadequate response to hydroxyurea. Ruxolitinib causes a blockade of the JAK-STAT pathway which is, among a lot of other effects, known to play a role in T effector cell responses , 6. InteMain severe side effects of ruxolitinib therapy, particularly in combination with other immunosuppressive therapies, are prolonged pancytopenia attended by severe infections and bleeding complications .Another therapeutical approach for aGVHD and cGVHD is extracorporeal photopheresis (ECP) using UV-A light in combination with 8-methoxypsoralen to induce apoptosis of leukapheresis gained mononuclear cells. Safety and efficacy of ECP administered in 21 patients with aGVHD and 88 patients with cGVHD as second- or third-line treatment in GVHD were reported recently. ORR in aGVHD was 84% .Here, we investigated a single-center experience of the tolerability and efficacy of combining both therapeutic strategies—treatment with ruxolitinib in combination with ECP—in 18 patients with steroid refractory acute GVHD of lower GI-tract after ASCT. Endpoints of this study were complete remission of steroid refractory aGVHD, duration of response, incidence of severe infections, cytopenias and bleeding complications, as well as 2 year overall survival.r: 21–73), stem cell source was in all cases peripheral blood. Steroid refractoriness was defined as no improvement in 7 days or aggravation after 5 days of steroid treatment with 2 mg methylprednisolone/kg bodyweight.From June 2015 to February 2017, 18 patients  with steroid refractory aGVHD of lower GI-tract who underwent an ASCT at the Department of Stem Cell Transplantation at the University Medical Center Hamburg-Eppendorf, Germany, due to different underlying diseases were treated with ruxolitinib and ECP (Table n = 7), liver (n = 6), or upper GI-tract (n = 2) or a combination of them and all patients showed overall grade III (50%) or IV aGVHD (50%) at starting ruxolitinib or ECP therapy according to classification system of Przepiorka et al. [Some patients showed additional aGVHD of skin  received ruxolitinib before starting ECP with a median interval between starting ruxolitinib and initiation of ECP therapy of 19.7 days (r: 7–62). Median day of onset of any grade of any aGVHD after transplantation was day 30 (r: 11–198), median start of ruxolitinib or ECP was day 86.5 (r: 35–257), but prompt after fulfilling definition of steroid refractoriness. Dosage of methylprednisolone in all patients at initiation of ruxolitinib or ECP was 2 mg/kg bodyweight. Median duration of ruxolitinib therapy was 59.8 days (r: 14–192) with a median start dosage of 20 mg per day . All patients started with two ECP treatments per week for two weeks with an individual reduction of treatment frequency, but in majority of patients, following ECP treatment was administered one time a week (r: 0.5–2.1 ECP treatments/week). Number of all ECP treatments differed from 2 to 71 treatments per patients (median: 20.5 treatments per patient), accordingly the duration of treatment differed from 0.4 to 23.4 months (median: 5.7 months). All ECP were performed with Therakos Cellex System.The majority of patients  status of all patients as an indicator for infections and immune status of ten patients (lymphocyte count with CD4 + T lymphocyte and regulatory T cell count) were collected prior and after four weeks of combined treatment with ruxolitinib and ECP and 3–4 weeks after stopping ruxolitinib therapy. Regulatory T cell population was identified by sequential gating for SSC low and CD45 dim cells with CD3, CD4, and CD25 positivity. Fourteen patients (78%) were CMV-positive before ASCT and 12 patients (67%) received stem cells of CMV-positive donors. Three of these patients had high-risk constellation for suffering of CMV reactivation being CMV-positive before ASCT and receiving CMV-negative stem cells.n = 10), including complete remission of 44% (n = 8) and partial remission of 11% (n = 2), while eight patients (44%) finally showed no response to the treatment. Response criteria were determined according to Przepiorka et al. [Overall response to the treatment with ruxolitinib, the combined immunosuppressive therapies and ECP in all patients were 56% , followed by receiving complete remission status , severe infections or sepsis , and in three patients (17%) no response to ruxolitinib and ECP therapy was the reason to stop the therapy . In 33% of patients (n = 6), ruxolitinib and ECP therapy had to be discontinued for a few days (r: 1–10) due to different reasons , but all of the patients could restart with the therapy with same dosage as previously given. In the group of the eight patients who stopped ruxolitinib and ECP treatment due to cytopenia, three patients could recover platelet, hemoglobin, and leukocyte level within four weeks, two patients died in this cohort.The main reason for stopping either ruxolitinib or ECP therapy was cytopenia  side effects associated with ruxolitinib and ECP could be observed, just in one patient no side effects have been documented Table . As expen = 12); all of the three patients mentioned in “Patients and methods” with high-risk constellation of host CMV positivity before ASCT, receiving CMV-negative donor stem cells were in this group of CMV reactivations after transplantation.Increase of CRP level (C-reactive protein) over 50 mg/dl could be observed in nine patients (50%), three of them developed a sepsis. Fever was observed in eight patients (44%). CMV reactivation during ruxolitinib therapy as a parameter of infection occurred in 67% of cases (p = 0.02) and after stopping treatment, regulatory T cell count decreased again . Complete stopping of steroids could be reached in a median time of 27 days after starting ruxolitinib and ECP therapy . Reduction to a quarter of initial dosage of methylprednisolone was reached in a median time of 6 days  with a median estimated overall survival of 15.3 months. Comparing patients who did not respond to the therapy to those who reached CR or PR, a lower overall survival of 38% in non-responders  could be observed than in the patients with CR/PR with an estimated overall survival of 70%  days of the ten surviving patients after 2 years after starting ruxolitinib and ECP therapy, five patients had continuous CR of aGVHD, four had continuous PR, and just one patient had a relapse of aGVHD. Overall, cGVHD developed in 67% (n = 12). Two patients developed severe cGVHD, four patients moderate, and the majority of six patients suffered from mild cGVHD according to NIH classification [After a median follow up of 886.9 (fication . Just twIn this single-center study, only patients with severe lower GI steroid refractory aGVHD—defined by no improvement in 7 days or aggravation after 5 days of steroid treatment with 2 mg methylprednisolone/kg bodyweight—were included. All patients were treated in a combined approach using ruxolitinib and ECP in order to increase the rate of responders and to show an increase of regulatory T cells. Treatment of severe steroid refractory aGVHD is still challenging in clinical practice. Finding a balance between therapy and side effects in this special cohort of patients is the main difficulty. On the one hand, therapeutic strategies with high efficacy are necessary to reduce symptoms and repress this complication after ASCT. On the other hand, it is important to keep the immunosuppressive effect as low as possible in order to reduce severe infections caused by an affected host immune system and to maintain the graft-versus-leukemia effect. Ruxolitinib and ECP are well known to show efficacy in prevention and treatment of acute and cGVHD in preclinical and clinical settings –11, 13. Our cohort represents severely affected patients with steroid refractory acute GVHD overall grades III or IV. Combining ruxolitinib and ECP, we could show a high rate of complete remission of 44%, a partial remission rate of 11%, and an encouraging 2 year overall survival of 70% in those patients who could reach CR or PR. In previous study, ECP alone could show high rates of partial remission, but very low rates of complete remission in steroid refractory aGVHD of 4.8% . HoweverAnalyzing immune reconstitution in selected patients we could show that regulatory T cells increased during the treatment of ruxolitinib and ECP, an effect which strengthen the assumption of potential efficacy of these two approaches for treating severe steroid refractory aGVHD, because it is well described that regulatory T cells can prevent graft-versus-host disease without negatively affecting graft-versus-leukemia effect , 15, 17.It is, in addition, remarkable that just one patient developed a relapse of aGVHD after stopping the combined therapy, perhaps underlying long-lasting effects of regulatory T cells induced by ruxolitinib therapy. However, developing cGVHD still remains a problem: high percentage of 70% developed cGVHD later on.In conclusion, we could show that combining ruxolitinib and ECP in severe steroid refractory aGVHD after ASCT shows comparatively high efficacy and acceptable tolerability, allowing rapid tapering of steroids with a low rate of severe infections.


Sensory hair cells of mutants show disorganised hair bundles with abnormally thin stereocilia. The origin of this mutation is the insertion of a transgene which disrupts expression of the Grxcr1 (glutaredoxin cysteine rich 1) gene. We report here that Grxcr1 exons and transcript sequences are not affected by the transgene insertion in tde homozygous (tde/tde) mice. Furthermore, 5’RACE PCR experiments showed the presence of two different transcripts of the Grxcr1 gene, expressed in both tde/tde and in wild-type controls. However, quantitative analysis of Grxcr1 transcripts revealed a significantly decreased mRNA level in tde/tde mice. The key stereociliary proteins ESPN, MYO7A, EPS8 and PTPRQ were distributed in hair bundles of homozygous tde mutants in a similar pattern compared with control mice. We found that the abnormal morphology of the stereociliary bundle was associated with a reduction in the size and Ca2+-sensitivity of the mechanoelectrical transducer (MET) current. We propose that GRXCR1 is key for the normal growth of the stereociliary bundle prior to the onset of hearing, and in its absence hair cells are unable to mature into fully functional sensory receptors.Tasmanian devil ( Hair cells are specialized sensory receptors responsible for the conversion of mechanical stimuli into electrical signals, which are relayed to the central auditory system via the afferent nerve fibres. Hair bundles protrude from their apical surfaces. The hair bundle is composed of rows of microvilli-like elements called stereocilia, which are graded in height to form a staircase-like structure . MechanoGrxcr1 gene encodes glutaredoxin cysteine rich 1, GRXCR1, which has been shown to target actin-rich structures in cultured cells and is mainly localized at the stereociliary bundles in cochlear and vestibular hair cells  (eqn.2), where k is a proportionality constant, Vr is the reversal potential, Vs is a measure for the steepness of the rectification, and γ is the fractional distance within the membrane’s electrical field of an energy barrier, as measured from the outside.Mechanoelectrical transducer (MET) currents were elicited by stimulating the hair bundles of OHCs using a fluid jet from a pipette (tip diameter 8–10 μm) driven by a piezoelectric disc). The pipette tip of the fluid jet was positioned near to the bundles to elicit a maximal MET current. Mechanical stimuli were applied as force-steps or saturating 50 Hz sinusoids  with driving voltages of ± 40 V. Membrane potentials were corrected for the liquid junction potential (Cs-based intracellular solution: –11 mV). Voltage clamp protocols are referred to a holding potential of –81 mV. The peak MET current-voltage curves were fitted according to a simple single-energy-barrier model : I(V) = t-test. Mean values are quoted ± s.e.m. where P < 0.05 indicates statistical significance.Statistical comparisons of means were made by Student’s two-tailed tde) homozygous mutants display hyperactivity, head-bobbing and circling, indicative of vestibular defects, and they lack a Preyer reflex (ear flick in response to noise) and cochlear responses to sound suggesting a severe hearing impairment. Scanning electron microscopy of the organ of Corti at P7 confirmed that the stereocilia of tde homozygous (tde/tde) mice were abnormally thin with a flaccid and disorganized appearance in both inner hair cells (IHC) and outer hair cells (OHC) compared to control heterozygous (+/tde) mice  mice .http://umgear.org) to look at Grxcr1 expression in single cell and bulk RNAseq datasets at P0 [Grxcr1 mRNA levels in the inner ear of tde/tde and control littermates at P7. We found that the expression of Grxcr1 was almost completely abolished in tde/tde inner ears at P7 .GRXCR1 has previously been shown to be specifically expressed in the cochlea and localized to the neuroepithelium containing hair cells and supporting cells of pre-hearing mice at P5 . We usedts at P0 , 30 and ts at P0  and founGrxcr1 [Grxcr1 in tde mutant mice, we performed PCR analysis and subsequent sequencing of the amplified bands (see Table 1 for the primers). One transcript of Grxcr1 is described in Ensembl: Grxcr1-201 (ENSMUST00000094715); 1,071 base pairs in 4 exons), which is translated into a protein of 296 amino acids . We analysed the genomic sequence of all four coding exons of the Grxcr1 gene using primers specific for each exon (Table 1), covering the entire coding region and the splice sites, using genomic DNA from phenotyped adults (n = 1 +/+ (C57BL/6N); n = 12 +/tde; n = 16 tde/tde mice). Exons were sequenced by capillary sequencing and traces were analysed. No changes were found in the coding sequence of any exon of the Grxcr1 gene in the homozygous mutants compared to wild-type C57BL/6N and +/tde mice  show severe hair bundle disruption and abnormally elongated stereocilia [tde/tde mice. The pattern of expression of MYO7A in the hair cells was broadly similar in tde/tde and control mice, with labelling evenly distributed within the top of the hair cell .MYO7A is an unconventional myosin expressed in sensory hair cells and stereocilia, and known to be involved in developing the correct hair bundle architecture , 35. Micreocilia , 36, whiair cell .Eps8 mutant mice have shorter and more numerous stereocilia than those of control mice [tde/tde mutants and littermate controls.EPS8 is an evolutionarily conserved signal transducer known to have multiple functions in the control of actin dynamics. rol mice . The locPtprq lead to different degrees of hair bundle disorganization and formation of giant stereocilia, and hearing loss [tde/tde mice [tde/tde mice was generally similar to that of littermate controls  current was recorded from P7 apical-coil OHCs by displacing their hair bundles using a piezo-driven fluid-jet. Upon moving the bundles in the excitatory direction  and at negative membrane potentials, a large inward MET current could be elicited in OHCs from both control (+/+) and mutant (tde/tde) mice (n = 4) compared to control  mice. Any resting current flowing through open MET channels in the absence of mechanical stimulation was reduced when bundles were moved in the inhibitory direction  in all control and mutant OHCs  compared to control  mice. Because the MET current reverses near 0 mV, it became outward when excitatory bundle stimulation was applied during voltage steps positive to its reversal potential 2+ influx [We then investigated whether the abnormal hair bundles of ant OHCs , arrows.+ influx , 41.We then investigated whether the adaptation properties of the MET current were affected in Tasmanian devil mice by stimulating the hair bundles of OHCs using mechanical step stimuli instead of sinusoids. Excitatory bundle movements with non-saturating stimuli elicited rapid inward currents at a holding potential of –81 mV that declined or adapted over time in both control and mutant P7 OHCs , arrows.2+ sensitivity of the transducer apparatus by deflecting the bundle in the presence of an endolymph-like solution containing 0.02–0.04 mM Ca2+ [2+ influx into the transducer channel by either depolarizing hair cells to near the Ca2+ equilibrium potential  in both control and mutant OHCs, the increment was much larger in the former  but not in mutant cells  control (+/+) IHCs were similar to those in tde/tde cells (Table 2). Moreover, all immature IHCs investigated were able to generate spontaneous repetitive Ca2+ action potentials  as previously shown in wild-type cells [tde/tde mice. We then investigated whether IHCs were able to acquire functional maturity, which is associated with the down-regulation of immature ion channels and acquisition of adult-like electrical properties including a rapidly activating large conductance Ca2+-activated K+ current  and a current carried by KCNQ4 channels  with an unusually hyperpolarized activation range. Potassium currents in IHCs were elicited in response to depolarizing voltage steps in 10 mV increments from –154 mV . Both IK,f and IK,n were present in adult IHCs (P20-P21) from control  by P14-P16 , 50. Thechannels , 52. In tde mutants is likely to be the consequence, not the cause, of reduced transduction current by analogy with other recent observations showing that defects in the MET current directly influence IHC development [We suggest that the immature state of IHCs in elopment .+ currents, the development of ICa and the induced exocytosis fail to mature in tde/tde mice. Exocytosis was estimated by measuring increases in cell membrane capacitance (ΔCm) following depolarizing voltage steps, which is generally interpreted as an indication of neurotransmitter release from presynaptic cells. The synaptic machinery of IHCs becomes more sensitive to Ca2+ as they mature, causing synaptic vesicles to be released linearly with increases in Ca2+ current. We found that in IHCs from tde/tde mice the developmental linearization of the exocytotic Ca2+ sensitivity did not occur. In adult tde/tde IHCs the maximal size of the Ca2+ current (ICa) was significantly larger (P<0.0005) than that of control cells  than in control adult IHCs  and Kcnq4, and both showed apparently reduced immunolabelling  . The resting membrane potential of mature OHCs was significantly more depolarized in mutant than in control cells  . Any redtde mice .tde/tde) homozygous mutant mice, in which Grxcr1 is severely down-regulated in the inner ear, we found that the expression patterns of four key proteins involved in the elongation and widening of the stereocilia  was generally similar in mutants compared with controls with normal hair bundles. We have demonstrated that mechanoelectrical transducer (MET) currents could still be recorded in pre-hearing OHCs from tde/tde mice despite the presence of abnormal stereociliary bundles. However, the MET current was smaller than in littermate controls, with a reduced resting open probability. Both IHCs and OHCs of tde/tde mice were unable to become fully functional sensory receptors after the onset of hearing, since they retained the basolateral membrane protein profile of immature cells. We conclude that GRXCR1 plays an essential role in the development of the hair bundle and regulates the morphological and function development of mammalian cochlear hair cells.We have shown that GRXCR1 is crucial for the growth of the stereociliary bundles in cochlear hair cells, and is required for the functional maturation of these cells into sensory receptors. Despite the thin stereocilia and disorganised hair bundles in the Tasmanian devil  mice the same intron is affected [Grxcr1 expression. Low amounts of mRNA suggest residual GRXCR1 protein might be expressed in hair cells, but not enough to maintain normal function.In affected , suggesttde/tde mice caused a largely reduced size of the MET current and of the resting open probability (Po) of the MET channels. While the smaller MET current could be caused by the loss of tip links between the disorganized stereocilia, the reduced resting Po and its Ca2+ sensitivity are most likely caused by a reduced tension in the remaining tip links. A reduced MET channel Po has also been reported in several mouse models affecting the bundle structure  or Ca2+ (Atp2b2 ). The in  have abnormally thin and long stereocilia, and their hair bundles become progressively more disorganized with age [Grxcr1 zebrafish mutants [Gene expression and immunostaining assays have shown that GRXCR1 is present in the sensory hair cells of the inner ear from early stages of development onwards, and localizes in the stereociliary bundle . Cochleawith age , 14. Sim mutants . Despite mutants , 14, ind mutants . Changes mutants .Eps8  encodes8  to exon 4 (bold), including the entirety of exon 3 (underlined), and lacking exon 2.5’RACE PCR sequence of (DOCX)Click here for additional data file.


Prunus salicina, ‘Black Golden’ Japanese Plum, during 35 days of cold storage followed by 3 days at 24 °C to evaluate its shelf life. Chitosan coating delayed the physico-chemical traits such as the total soluble solids, titratable acidity, firmness and skin color associated with the plum ripening process during cold storage and shelf life. Furthermore, the highest bioactive compounds’ content and antioxidant activity in the coated plum were registered during the stored period. Chitosan-based coating enhanced the activities of superoxide dismutase and ascorbate peroxidase during cold storage and shelf-life. Moreover, this edible coating contributes to reducing membrane damages with lower lipoxygenase activity and, consequently, malondialdehyde accumulation. A multivariate statistical analysis approach identified the two key components, correlated to all analyzed traits, that influenced the changes within chitosan coated and uncoated Japanese plums during storage. Chitosan coating is a cheap and eco-friendly tool to delay ripening process and to improve the antioxidant systems and storability of the ‘Black Golden’ Japanese plum.In this study, the influence of a chitosan-based coating was assessed on qualitative traits and non-enzymatic and enzymatic antioxidant systems in  Prunus salicina Lindl.) is an important stone fruit species cultivated worldwide, with a global production that reaches over 12 million tons [Japanese plum , owned by the CREA Center of Research For Olive, Fruit and Citrus Crops . Fruit were transported to the laboratory and screened for uniform size and the absence of defects or decay.w/v) was realized as described by Adiletta et al. [w/v) chitosan concentrations (data not shown). Fruit were stored at 2 °C and 95% relative humidity and were analyzed every week until 35 days of cold storage (CS) and subsequently they were evaluated after 3 days of shelf life (SL) at 24 °C Uncoated fruit were dipped in distillate water. Five biological replicates per treatment (eight fruit per replicate) for each sampling date were prepared. Three replicates were used to assess the fruit properties during cold storage, while the other two were used for testing the SL. All analyses were assessed at harvest, at each sampling date and after SL.Fruit were randomly distributed into two lots (approximately 250 fruit in each lot). Chitosan solution with 90% deacetylation and a molecular weight of 360 kDa at 1%  were homogenized with methanol (80% v/v) containing 2 mM NaF; the extract was centrifuged at 15,000× g for 15 min at 4 °C. The Folin–Ciocalteu method was used to quantify the total phenolics (TP) using gallic acid as standard and results were expressed as mg gallic acid equivalent (GAE) per 100 g fresh weight (FW) [Total phenolics, anthocyanins, flavonoids and antioxidant activity were determined using methanolic extract completed in accordance with Martínez-Esplá et al. . Fruit fght (FW) . The totght (FW)  and the ght (FW) . The resght (FW) .v/v) metaphosphoric acid and  diluted Folin’s reagent, in a final volume of 2 mL. The results were expressed as mg ascorbic acid (AA) per 100 g FW.The ascorbic acid content (AA) of the plum fruit was determined following the method described by Petriccione et al.  with a mw/v) in 100 mM potassium phosphate buffer (pH 7.8) containing 1 mM sodium EDTA (pH 7), 2 mM DTT, 1 mM PMSF, 0.2% Triton X-100, 5% (w/v) PVPP, and 5 mM ascorbic acid (the ascorbic acid must be used only for APX enzyme extraction), as reported by Adiletta et al. [Crude enzyme extract was prepared by homogenizing frozen fruit powder  and 150 µL of crude enzyme extract in a final volume of 1.5 mL. APX activity was expressed in µmol of ascorbate per mg protein. Superoxide dismutase  activity was assessed as reported by Adiletta et al. [Ascorbate peroxidase  activity was evaluated according to Modesti et al.  monitoria et al.  based on2O2, 6.4 mM guaiacol and 250 µL of crude enzyme extract in a final volume of 1 mL. Polyphenol oxidase (PPO) and lipoxygenase (LOX) activity were determined followed the method and the assay buffers described by Pasquariello et al. [Guaiacol peroxidase  activity was assayed according to Modesti et al. . The reao et al. . PPO actGPX, PPO and LOX activity were expressed as mmol per mg protein and µmol per mg protein, respectively.Malondialdehyde (MDA) content was measured by the method of Adiletta et al. . Mixturep < 0.05 were considered significant and were indicated with different letters. A principal component analysis (PCA) was applied to describe the influences of chitosan treatment on physico-chemical and antioxidant systems and to identify the components responsible for the main variations in the dataset. All analyses were performed using the SPSS® software package, version 20.0 .All results were expressed as the mean ± standard deviation. Statistical significance among chitosan-coated and uncoated plums was analyzed by one-way analysis of variance (ANOVA), and Duncan’s test at 5% level was calculated to compare the differences between means. Furthermore, statistical significance between storage times was evaluated. Differences at Cumulative weight loss showed higher values in the uncoated fruit compared to chitosan-coated fruit with progression of the storage period. After 35 days of cold storage uncoated and chitosan-coated fruit reached a maximum weight loss of 6% and 4%, respectively a. ChitosFirmness values decreased under the cold storage conditions b and aftChitosan coating delayed the increase in TSS in coated plums compared to uncoated ones throughout the cold storage and shelf life . ChitosaTA content displayed the same trends with a constant decrease in every sampling date (coated and uncoated fruit) during the storage and shelf-life period. TA loss was 44% and 57.08% in coated and uncoated fruit from harvest to the end of cold storage while after shelf life a further loss of 24.69% and 36.4% in the same samples was registered.Results also showed significant effects of storage conditions and chitosan coating on color parameters such as L*, Chroma (C) and Hue angle (H). These parameters displayed a decrease at each sampling date during cold storage with significant differences among chitosan coated and uncoated samples.The content of polyphenols (TP) and flavonoids (TF) increased throughout cold storage and shelf life in all analyzed samples . ChitosaThe content of anthocyanins and carotenoids increased in plum fruit during cold storage and shelf life but untreated fruit resulted in significantly higher values than the coated ones. Maintenance at room temperature led to a reduction of carotenoids’ content compared with the cold-stored sample with a higher percentage reduction after 35 days of cold storage plus 3 days at 24 °C.In plums a decrease in ascorbic acid content has been observed but chitosan-treated fruit were generally characterized by higher values during cold storage and shelf life at room temperature.Antioxidant activity increased until day 35 in both samples, with higher significantly differences at day 7 and day 14 plus 3 days of self-life in coated fruit compared with the uncoated fruit, respectively.Antioxidant enzymes involved in ROS detoxification in plum fruit throughout cold storage and shelf life are shown in Treatment with chitosan reduced the GPX activity significantly, showing an increase during first 14 days of cold storage followed by a decrease until the end of the experiment. The same trend of GPX activity has been observed during shelf-life period with higher values in uncoated fruit. PPO activity increased rapidly as storage time progressed mostly in untreated samples. Chitosan coating inhibited PPO activity by about 25% more than control after 35 days of cold storage. A slight increase in PPO activity was observed, maintaining the same gap between the coated and uncoated fruit at the end of shelf life.During cold storage and shelf life the membrane damages in treated and untreated fruit were monitored by measuring LOX activity and MDA content . LOX actCell membrane permeability was evaluated by electrolyte leakage to monitor the physical injury to the cell membrane resulting from oxidative stress in chitosan-coated fruit during cold storage and shelf life . ElectroPCA analysis was performed to pinpoint the influence of the chitosan treatment on the variations of physico-chemical and nutraceutical traits, antioxidant and browning enzymes in plum fruit during cold storage  and shelWL, TSS, EL, TAC, MDA content, PPO and LOX were highly positively correlated with PC1 whereas firmness, TA, AA, APX, SOD and L*, C and H were negatively correlated. The variables positively correlated with PC2 were TP, TF, DPPH while GPX was negatively correlated . ChitosaJapanese plum is considered to be a climacteric fruit and rapid physiological and biochemical changes occur during its postharvest life leading to the alteration of physico-chemical traits and losses in functional compounds . Further2 production, that causes an inhibition of cell wall-degrading enzymes’ activity, such as polygalacturonase and pectin methyl esterase responsible for the softening process and as suggested by Manganaris et al. [Nowadays postharvest management aims to extend the fruits’ shelf life applying green and eco-friendly strategies along the supply chain. Chitosan-based coating represents a low-cost, eco-friendly method to control the fruit quality of whole and fresh-cut fruit along the postharvest value chain . Chitosas et al. . Peel cos et al. ,16,17.2 and CO2 transmission rate through the coating layer reducing the respiration rate of the coated fruit and preserving the ascorbic acid oxidation due to ascorbic-acid oxidase and PPO activity whose activities directly depend on O2 concentration [Bioactive compounds’ content, as well as polyphenol and flavonoids, increased gradually and continuously with the increase in storage period with higher values in the chitosan coated fruit, suggesting that chitosan treatment delays fruit senescence and enhances the phytochemical content and antioxidant activity during storage ,32. Furtntration . Retentintration ,11,34. SFruit ripening is a complex, genetically programmed process where non-enzymatic and enzymatic antioxidant systems play an important role to maintain reactive oxygen species at steady-state level avoiding oxidative stress. Furthermore, the generation of ROS in response to postharvest management such as cold storage and edible coatings in several fruit has been reported ,15,35,36The results of this study confirmed that chitosan coating improved the activity of enzymes involved in the antioxidant defense system such as SOD and APX. In a study of ‘Sanhuali’ Japanese plum fruit it was registered that combined ascorbic acid and chitosan coating increased the SOD, POD and CAT activity . FurtherBrowning is related to fruit senescence and it is due to the oxidation of phenolic substrates mediated by PPO or to the GPX activity that catalyzes single-electron oxidation of several antioxidant compounds in the presence of hydrogen peroxide . Loss of2 availability in the plum fruit. Chitosan-coated fruit showed significantly lower GPX activity, probably as a result of delayed cell membrane damage occurring in response to low temperatures and chitosan treatment.Chitosan treatment reduced the PPO activity due to a low OPostharvest oxidative stress causes the loss of membrane integrity, and MDA content and EL values are two key indicators to evaluate membrane injury that results from the lipid peroxidation process during storage . The maiThe reduction in skin and flesh color changes are due to the preservation of membrane integrity and separation of PPO and GPX enzymes from their phenolic substrates. Similarly inhibited GPX and PPO activities have been observed in response to alternative technologies to chitosan coating employed on different fruit to improve their postharvest life ,38,40,41Japanese plums due to their perishable nature have a limited shelf life. Edible coatings represent an effective postharvest technique to prolong shelf life of several fruit crops. In this study, chitosan coating formed a barrier on fruit surface, which delayed qualitative decay improving the physico-chemical traits and bioactive compounds content during cold storage and shelf life in Japanese plum fruit. Furthermore, it improves bioactive compounds content with antioxidant properties and enzymatic antioxidant defense system, and consequently it controls lipid peroxidation responsible for the loss of membrane integrity in the fruit tissues and enzymatic browning.Chitosan-based coating is an effective preservative tool to maintain the quality, acceptability and nutritive value and thus exhibiting a huge potential for extending the postharvest life of Japanese plums during cold storage. Future research should be focused on the industrial scale application of the natural coatings to satisfy consumer demands and to reduce food losses through the supply chain.


The distinction between cardiac aneurysms and diverticula can be very difficult by angiography. Left ventricular (LV) aneurysms usually occur following transmural myocardial infarction. On the other hand, cardiac diverticula are most commonly congenital. They are commonly detected by cardiac CT with a prevalence of 2.2%. Here we present a case of a 60-year-old male with the incidental finding of multiple LV aneurysms masquerading as diverticula in the setting of myocardial infarction with near normal coronary arteries. Moreover, this case highlights the limitation of coronary angiography in the diagnosis of myocardial infarction with no obstructive atherosclerosis (MINOCA). A 60-year-old male with cardiac risk factors in the form of smoking and premature family history of coronary artery disease and no previous cardiac history, who presented to our facility with acute onset of retrosternal chest pain lasting for 30 minutes with radiation to the left arm. He denied any other symptoms. His medications included suboxone and ibuprofen.On examination his temperature was 36.9°C, blood pressure 161/94 and heart rate 86 beats per minute and regular. His cardiorespiratory examination was within normal limits with no evidence of congestive heart failure.9/L, hemoglobin 168 g/L, platelet count 339 × 109/L, his high sensitivity troponin peaked at 2997 ng/L . He had normal kidney function and coagulation profile. His initial electrocardiogram showed sinus rhythm with left axis deviation and Q waves in leads V1 to V2 .His investigations showed a white count of 10.6 × 10He was admitted and managed as a case of non-ST elevation myocardial infarction. He then underwent an inpatient coronary angiogram, which demonstrated mild coronary artery disease (CAD) and 2 diverticula in the mid anterior wall and larger one in the apical wall on ventriculography (Video). An echocardiogram with contrast revealed an ejection fraction of 64% with no wall motion abnormalities and no diverticulum or aneurysm seen. He had a cardiac Computed Tomography (CT) with contrast which confirmed the presence of mild coronary artery disease with calcified and non-calcified atherosclerotic plaque in the left anterior descending artery (LAD) and possible distal occlusion of a small diagonal branch  with foc. Left ventricular aneurysms may occur due to various causes including congenital, inflammatory, and traumatic or may complicate acute coronary syndrome (ACS). The incidence of this complication following ACS is declining due to improved treatment options in the post-interventional era. In addition, the occurrence of multiple left ventricular aneurysms in the setting non-ST elevation myocardial infarction and near normal coronary arteries is uncommon.Left ventricular aneurysms are a rare malformation. A thin, scarred well-delineated wall that balloons outward characterizes them, while LV diverticula are outpouchings composed of endocardium, myocardium, and pericardium. Cardiac CT was instrumental in refuting this diagnosis however, and revealed small diagonal occlusion and a septal branch occlusion from the LAD which probably accounted for the 2 focal apical lateral and mid septal aneurysms respectively.In our case, the patient had met the diagnostic criteria for myocardial infarction given his elevated cardiac biomarkers and ischemic chest pain with his angiogram initially demonstrating mild CAD in addition to the presence of an apical diverticulum. As such the diagnosis of MINOCA was made. MINOCA is a syndrome characterized by the presence of myocardial infarction (MI) in the setting of normal or near normal coronary arteries on angiography with luminal stenosis ⩽50%. Causes of MINOCA are broadly divided into epicardial, microvascular dysfunction, and coronary artery embolismThe CMR proved to be useful in delineating the etiology of his MI demonstrating delayed focal subendocardial enhancement  in the tThis case highlights the limitation of coronary angiography in the diagnosis of MINOCA. In addition, it highlights the expanding role of cardiac CT and MR as superior modalities to echocardiography in the diagnosis and further characterization of left ventricular aneurysms. The presence of multiple focal LV aneurysms on LV angiography with mild CAD, should raise the suspicion of small vessel occlusive disease resulting in chronic micro-infarcts, which may account for this rare clinical entity.Click here for additional data file.Supplemental material, sj-pdf-1-cic-10.1177_11795468211006698 for An Unusual Case of Multiple Left Ventricular Aneurysms Masquerading as Diverticula in the Setting of Myocardial Infarction by Hussain Alzayer, Ahmad Alshatti and Akeel Alali in Clinical Medicine Insights: Cardiology


PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene  CFSs are specific regions of the genome that often fail to replicate before mitosis, which results in chromosome breakage and high mutation rates . SeveralIt has become apparent that breakage and mutations at CFSs are due to replication problems caused by transcription of extremely large genes located at CFSs . To undePRKN underlies its fragility  was amplified using VV47 and VV49. All primers were designed to facilitate directional cloning and they were synthesized by TAG Copenhagen. The amplified products were cloned into pCR2.1-TOPO (Invitrogen) and confirmed by Sanger sequencing (performed by Eurofins Genomics).All constructs generated in this study are listed in KpnI SalI fragment, the 3′ homology arm was inserted as a BamHI NotI fragment, and a resistance cassette (BSR or PURO) was inserted as a BamHI fragment. Finally, the 2YFP fragment was inserted as a XhoI SalI fragment into the SalI site. Correct orientation was confirmed by restriction digest. The resulting constructs were named pVV6 and pVV15 encoding puromycin (PURO) or blasticidin (BSR) resistance, respectively.The fragments for the PRKN 2YFP-tagging construct were then assembled in pBluescript (SK+). Specifically, the 5′ homology arm was inserted as a PRKN intron-7 deletion, genomic regions flanking gRNA Target Site 1 (TS1) and gRNA TS2 were first amplified by PCR templated by genomic DNA (gDNA) from DT40 cells to obtain homology arms.To construct the repair template for ApaI and BamHI restriction sites, respectively. Similarly, PCR was conducted on gDNA with primers 3′ fwd and 3′ rev adapted with BamHI and XbaI restriction sites, respectively, to create the 3′ homology arm extending 3′ of gRNA TS2. The homology arm fragments were subcloned into TOPO TA vectors  and sequenced (Eurofins Genomics).To create the 5′ homology arm extending 2 kb 5′ of gRNA TS1, PCR was conducted on gDNA with the primers 5′ fwd and 5′ rev adapted with ApaI-BamHI fragment. Subsequently, the 3′ homology arm was subcloned from the TOPO TA vector into the 5′homology arm-pBluescript as a XbaI-BamHI fragment. Finally, the BSR cassette fragment was cloned in as a BamHI fragment. The final construct was sequenced to confirm correct assembly (Eurofins Genomics).Then, the 5′ homology arm was subcloned from the TOPO TA vector into pBluescript (SK+) as an BbsI site. Correct integration was confirmed by sequencing (Eurofins Genomics). The constructs were named pX458 PRKN TS1 and pX458 PRKN TS2.The backbone for the Cas9/gRNA constructs was pX458 (Addgene). The expression of specific target gRNAs was obtained by annealing the oligonucleotides listed in 2.All DT40 cell lines used in this study are listed in TM (BioRad) with the settings 25 μF and 0.6 kV. Approximately 35 μg of linearized plasmid DNA was used for transfection with the 2YFP targeting construct. For deletion of PRKN intron 7, 20 million cells were transfected with 50 μg linearized repair template and 30 μg of each of the two Cas9/gRNA expression vectors . Transfection with Cas9/gRNA was transient.Transfections for targeted integration were performed by electroporation with Gene Pulser XcellFor transient expression of the Cre recombinase, 3.5 million cells were transfected with 15 μg plasmid DNA using the nucleofector system developed by Amaxa Biosystems GmbH .® 10 image cytometer (ChemoMetec A/S) was used. Cells were stained by Vybrant Ruby Stain  to exclude dead cells based on their DNA content. Only cells meeting the following criteria were included in the analysis: not in aggregate, circularity > 0.6, and with DNA content of viable cells.For quantification of fluorescently tagged protein levels, the XcytoPACRG mRNA levels, 1 μg RNA was pre-treated with DNase I (Fermentas) to remove gDNA according to manufacturer’s instructions in the GeneJET RNA Purification Kit (Thermo Fisher Scientific). cDNA was made using RevertAid Premium Reverse Transcriptase (Thermo Fisher Scientific) with random hexamers and oligo(dT) primers.Total RNA was isolated using the GeneJET RNA Purification Kit (Thermo Fisher Scientific). For RNA samples used for analysis of –ΔΔCt method  (1:200), Biotinylated rabbit anti-streptavidin (Rockland) (1:266) for biotin-labeled probe detection and mouse anti-digoxygenin FITC (Interchim) (1:50) and goat anti-mouse (Alexa 488) for digoxygenin-labeled probe detection.Slides with metaphase spreads were mounted with coverslips using 15 μl of mounting medium containing DAPI . Metaphase chromosomes were visualized on a widefield microscope  equipped with a 100× objective lens , a cooled CCD camera , differential interference contrast (DIC), and an illumination source .PRKN gene, which is highly fragile in our model cell line (PRKN endogenously tagged with a Venus-YFP (2YFP) tandem tag in a background where the non-fragile gene TOPBP1 was endogenously tagged with TFP on one of its three alleles. The resulting cell line thus has the genotype PRKNWT/2YFPTOPBP1WT/WT/TFP and is referred to as “P2Y-TT”. Following tagging of PRKN, the allele remained fragile in response to replication stress and the tagged gene product was expressed at full length  cassette. Flanking loxP sites enabled subsequent removal of the BSR cassette. Initial PCR screening suggested that some clones potentially had the 80 kb region replaced by the cassette . Thus, we examined the karyotype of the clones derived from this transfection for aneuploidy. Here, we found that 2 out of 14 clones were trisomic for chromosome 3  with two different primer sets: One set binding specifically to transcripts from the 2YFP-tagged allele (PRKN2YFP) and one set binding to transcripts from both the 2YFP-tagged and untagged (PRKNWT) allele   . Only tht levels  or totalt levels  were detPRKN. Specifically, we exploited that PRKN shares its promoter (marked by high GC content in PACRG (parkin coregulated)  , which icription . While aPRKN intron 7 does not markedly alter PRKN or PACRG expression.Altogether, this indicates that the 80-kb deletion in PRKN are underlying its fragility, we performed FISH on metaphase spreads from the two PRKNWT/Δin 7 clones after inducing replication stress by treatment with the DNA polymerase inhibitor aphidicolin (APH). These clones enabled us to use the full-length wild-type allele of PRKN as an internal control. For FISH, we used a probe that binds PRKN outside of intron 7, which detects both full-length and intron 7-deleted PRKN, as well as a probe that binds the deleted region of intron 7 and therefore only detects the full-length PRKN (PRKN locus in the PRKNWT/Δin 7 clones was as fragile as the full-length PRKN in the parental cell line, the intron 7-deleted PRKN allele was significantly less fragile in both PRKNWT/Δin 7 clones (PRKN, resulted in an approximately 50% reduction of the fragility of the gene. Thus, the 80 kb region in intron 7 clearly contributes to PRKN fragility even though this region does not have a clear role in PRKN expression or cell viability.To test the hypothesis that the large introns in gth PRKN . While t7 clones . NotablyPRKN gene, which is located in a highly fragile CFS (PRKN intron 7, clearly demonstrating that the deleted region is not essential for cell viability. Moreover, we find that this intron 7 truncation does not have any significant effect on PRKN gene expression. Yet, the 80-kb intronic deletion leads to an almost 50% reduction of PRKN fragility although only shortening the gene length by 12%, which does not appear to be a consequence of altered transcriptional activity. Thus, these 80 kb of intronic sequence with no apparent function are significantly contributing to PRKN fragility, most likely reflecting that extreme gene size is a trigger for chromosomal fragility, which suggests a disproportional significance of gene length on chromosomal fragility. The reason for the reduction of fragility upon intron deletion might be that conflicts between transcription and replication are avoided due to the shorter traveling time for the RNA polymerase. However, given the importance of replication timing for fragility, the reduced fragility may result from change in replication timing, which we expect to occur because the transcription unit is shortened and thereby the distance between replication origins at each side of the gene will be reduced. However, further studies are needed to experimentally determine the effect of intron deletion on replication timing. We note that the size of the gene or elements within the intron may play a functional role in certain tissues. It may even be possible that genomic instability at CFSs play a physiological role for instance in neurons where it might serve to generate genetic diversity (Extremely large genes arose in an early vertebrate ancestor due to intron expansions . Furthergile CFS . This isiversity . Alternaiversity .Here, we find that intronic truncation does not lead to changes in gene expression, thus adding to the mystery regarding the conservation of large introns. Therefore, further studies are needed to unravel the functional significance of large introns in genes at CFSs that clearly cause problems for dividing cells.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the animal study because the DT40 parental bursal lymphoblast cell line is commercially available from ATCC.SM performed the 80-kb deletion of PRKN intron 7 and most of the experiments that relate to this. VV constructed the cell lines expressing tagged parkin and generated the intron 7 FISH probe, and also assisted with supervision and data analysis. VO conceived, coordinated, and supervised the project. VO, SM, and VV wrote the manuscript. All authors contributed to the article and approved the submitted version.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. The reviewer VB declared a shared affiliation with one of the authors SM to the handling editor at the time of review.


An increase in the average global temperature and drought is anticipated in sub-Saharan Africa (SSA) as a result of climate change. Therefore, early white quality protein maize (QPM) hybrids with tolerance to combined drought and heat stress (CDHS) as well as low soil nitrogen (low-nitrogen) have the potential to mitigate the adverse effects of climate change. Ninety-six early QPM hybrids and four checks were evaluated in Nigeria for two years under CDHS, low-nitrogen, and in optimal environments. The objectives of this study were to determine the gene action conditioning grain yield, assess the performance of the early QPM inbred lines and identify high yielding and stable QPM hybrids under CDHS, low-nitrogen and optimal environment conditions. There was preponderance of the non-additive gene action over the additive in the inheritance of grain yield under CDHS environment conditions, while additive gene action was more important for grain yield in a low-nitrogen environment. TZEQI 6 was confirmed as an inbred tester under low N while TZEQI 113 × TZEQI 6 was identified as a single-cross tester under low-nitrogen environments. Plant and ear aspects were the primary contributors to grain yield under CDHS and low-nitrogen environments. TZEQI 6 × TZEQI 228 and the check TZEQI 39 × TZEQI 44 were the highest yielding under each stress environment and across environments. Hybrid TZEQI 210 × TZEQI 188 was the most stable across environments and should be tested on-farm and commercialized in SSA. Zea mays L.) is an economically important annual cereal crop that is predicted to become the most important cereal crop in the world by 2025 [Maize  and commercialized in West and Central Africa (WCA) [According to Neate , 40% of ca (WCA) . Howeverca (WCA)  highlighMaize breeding efforts in recent years have recorded significant successes in identifying lines that could be used as tolerance donors for breeding of cultivars with CDHS tolerance. Cairns et al.  identifiBreeding of early white QPM maize hybrids with tolerance to CDHS and the information on the gene action involved in the inheritance, the combining ability and heterotic patterns of inbred lines in the IITA-MIP are crucial to their successful use for hybrid development and production ,28. A reThe availability and use of early white QPM hybrids with tolerance to CDHS and low-nitrogen conditions would be more sustainable for smallholder farmers in nitrogen-depleted areas of SSA where low levels of agricultural inputs are utilized and there is simultaneous incidence of drought and heat stresses . Therefop < 0.01) variations among environment (E) and genotype (G) mean squares for the measured traits except for E mean squares for plant height and G mean squares for anthesis-silking interval (ASI) and plant height. Significant differences for genotype by environment interaction (GEI) mean squares were also observed for measured traits except for plant and ear heights  revealed significant ( heights . PartitiP < 0.01) differences in mean squares for E, G and GEI for grain yield and other measured traits except G mean squares for ASI and plant aspect and GEI mean squares for ASI  differences in mean squares were observed for E, G and GEI for the measured traits, except the G and GEI mean squares for grain yield and ear rot (p < 0.01) were observed for GCAm, GCAf and SCA mean squares for measured traits except GCAf and SCA mean squares for grain yield and ear rot. Furthermore, significant mean squares for GCAm × E, GCAf × E and SCA × E were observed for measured traits except for grain yield. Broad-sense heritability estimates varied from 13% for ears per plant to 78% for plant height while grain yield recorded H2 of 41%. Moderate to high repeatability estimates were recorded for measured traits. Across research environments, significant variations (p < 0.01) were observed among E, G and GEI mean squares for measured traits except the GEI for plant height  among the parental lines  with a mean of 67% while grain yield reduction under low-nitrogen environments revealed a yield reduction range of 8% to 74% with a mean of 44.9%. Overall, the CDHS and low-nitrogen tolerant QPM hybrids recorded lower reductions in grain yield than the susceptible QPM hybrids  biplot procedure. The GGE biplot analysis revealed that the first and second principal component axes explained 65.5% of the total variation in grain yield of the QPM hybrids . The “whThe “mean vs. stability” of the GGE biplot view was used to identify the highest yielding and the most stable QPM hybrids across the test environments . The verUnder the low-nitrogen environment, plant and ear aspects were identified by the stepwise multiple regression analysis as the first order traits, with significant contributions to grain yield and explaining 73% of the total variation in the grain yield of the QPM hybrids . Plant ap < 0.01) observed among the early QPM hybrids under low-nitrogen, CDHS, optimal and across test environments indicated that considerable genetic variation existed among the genotypes to facilitate accelerated gains from selection under contrasting environments. The observed significant differences for environments among the QPM hybrids revealed that each research environment was unique in terms of climatic and edaphic conditions which further suggested the need for extensive evaluations in contrasting environments. The significant genotype by environment interactions observed for grain yield and other measured traits under low-nitrogen, CDHS, optimal environments and across test environments implied that the hybrids responded distinctively to the environments. This emphasized that the environments were different and that the performance of the hybrids would not be consistent in different environments. Similar findings were reported by Ertiro et al. [The significant genotypic variation , GCAf × environment (E) and SCA × environment (E) interactions observed for measured traits except the GCAm × E for ASI and the GCAf × E for grain yield under low-nitrogen conditions, indicated that the performance of the parental lines were consistent across the low-nitrogen environments. This result is in agreement with the findings of Derera et al.  and OyekUnder low-nitrogen environments, comparable contributions of GCAm and GCAf sum of squares were observed for grain yield and other measured traits except ASI, indicating that both maternal and paternal effects were equally important in the inheritance of the measured traits of the hybrids. Similar studies by Ifie et al. , Annor aAccording to Girma et al. , the infUsing the HGCAMT grouping method, the 24 QPM inbred lines were classified into four heterotic groups. Interestingly, crosses between inbred lines from different heterotic groups displayed higher heterosis across stress environments indicating the effectiveness of the grouping method. The heterotic groups identified by the HGCAMT method across environments would increase the chances of developing outstanding early maturing QPM hybrids and synthetics with CDHS and low-nitrogen tolerance for commercialization in SSA. Additionally, heterotic populations could be developed by recombining QPM inbred lines from the same heterotic group and improving the population through recurrent selection methods .Based on the criteria proposed by Pswarayi and Vivek  for idenAn important objective of this study was to identify the most outstanding hybrids under each and across test environment conditions. The QPM hybrids TZEQI 6 × TZEQI 219 and TZEQI 6 × TZEQI 228 were identified as the most outstanding across CDHS and low-nitrogen environments. These hybrids could be useful in the development of early maturing multiple-stress tolerant three-way QPM hybrids for commercialization in SSA. The significant genotype by environment interaction observed for grain yield under CDHS, low-nitrogen and across test environments in the present study justified the use of the GGE biplot to identify high yielding and stable QPM hybrids for commercialization in SSA. From the “which-won-where” view of the GGE biplot, hybrids TZEQI 241 × TZEQI 216, TZEQI 6 × TZEQI 210, TZEQI 39 × TZEQI 44 (check) and TZEQI 6 × TZEQI 55 (check) were identified as the highest yielding in low-nitrogen, optimal and CDHS environments, respectively. Additionally, the mean yield vs. stability of the GGE biplot view identified TZEQI 39 × TZEQI 44 (check) as the highest yielding and TZEQI 210 × TZEQI 188 as the most stable QPM hybrids across test environments. These results suggested that these hybrids would display superior performance in nitrogen depleted, drought prone environments without compromising grain yield.Because of the quantitative nature of grain yield and the low heritability recorded for grain yield under the stress environment conditions used in this study, selection using grain yield alone without other agronomic traits would be ineffective . Therefo1 maize hybrids of ten bi-parental crosses involving crosses among extra-early white QPM inbred testers and early maturing white QPM inbred testers. The QPM inbred line testers were identified to have positive and significant general combining ability from previous studies [1 hybrids were taken through a cycle of backcrossing to the extra-early inbred testers to recover the earliness. The BC1F1 with desirable agronomic characteristics were selected using pedigree selection from each backcrossed population and advanced through repeated inbreeding to the S7 generation while selecting for kernels with the appropriate endosperm modification ranging from 25–50%  and 15 kg N ha−1 were applied at 2 WAP with additional 15kg N ha−1 applied at 4 WAP. The low-nitrogen plots received 60 kg ha−1 each of single superphosphate (P2O5) and muriate of potash (K2O) at 2 WAP. The low-nitrogen plots were kept weed-free with the application of atrazine and gramozone as pre- and post-emergence herbicides at 5 L/ha, respectively, and subsequently supplemented with hand weeding to keep the plots weed-free.The 96 early white QPM hybrids generated from the crosses of the 24 QPM inbred lines and four released IITA commercial hybrid checks  were evaThe 96 early white QPM hybrids plus four hybrid checks were also evaluated for their agronomic performance under CDHS conditions at the IITA experimental station in Kadawa  during the 2020 and 2021 dry seasons, where extreme drought stress at high temperatures between 33 and 45 °C occurred between February and June every year [−1, 60 kg ha−1 each of P2O5 and K2O at 2 WAP and additional 60 kg ha−1 N was top-dressed using urea at 4 WAP. The trials were kept weed free as described in the other trials.The QPM hybrids were also evaluated using 10 × 10 lattice design with two replications respectively under optimal conditions at Mokwa and Kadawa during the 2019/2020 (between June and October) rainy seasons. Fertilizer was applied at the rate of 30 kg N haData were collected under each research conditions for number of days to 50% silking (DS) and anthesis (DA). Anthesis-silking interval (ASI) was calculated as the difference between DS and DA. Plant height (PLHT) was measured as the distance from the base of the plant to the height of the first tassel branch. Ear height (EHT) was measured in centimetres as the distance from the base of the plant to the node bearing the upper ear. Root lodging (RL) was measured as the percentage of plants leaning more than 30°(degree) from the vertical whereas stalk lodging (SL) was recorded as the percentage of plants broken at or below the highest ear node. Plant aspect (PASP) was recorded on a scale of 1 to 9 based on plants appeal to sight, where 1 = excellent phenotypic appeal and 9 = extremely poor phenotypic appeal. Ear aspect (EASP) assessed the freedom from disease and insect damage; ear size and uniformity of ears was scored on a scale of 1 to 9, where 1 = large, uniform, clean, and well-filled ears and 9 = ears with totally unattractive features. Number of ears per plant (EPP) was calculated by dividing the total number of ears harvested per plot (EHARV) by the number of plants in a plot at harvest (PHARV). Ear rot (EROT) was computed as the percentage of ears harvested that had ear rot. Stay green (STGR) characteristic was scored at 70 DAP on a 1 to 9 scale as percentage of dead leaf area under low-nitrogen and CDHS conditions, where, 1= all leaves are green and 9 = all leaves are dead. Data on tassel blast (TABLAST) and leaf firing were recorded during flowering on the CDHS trial. TABLAST was rated during flowering on a scale of 1 to 9, where 1 = all plants had normal pollen production and 9 = all plants had white, dry tassels without pollen production and showed severe tassel blast. 2) estimates for each measured trait was calculated for each measured traits under each environment, and across the environments were estimated as follows:r is the number of replication and e is the number of environments.Data obtained for grain yield and other measured agronomic traits under low-nitrogen and CDHS environments were subjected to analysis of variance (ANOVA) with PROC GLM statement in SAS version 9.4  to obtaiironment . Repeataironment . Thus, rf and Xm were the mean performance of the male and female parents respectively and µ is the overall mean of crosses in the trial. The proportionate contribution for each measured trait was calculated as the percentage of the sum of squares for the crosses attributed to general combining ability (GCA) and specific combining ability (SCA). The general combining ability (GCA) effects for male and female within sets (GCAm and GCAf) for each measured trait were computed from the adjusted means using line × tester approach . As showfe, and MSme were the mean squares of the interactions between male and female with the environment; MSfme = was the mean squares of the female × male by environment interaction. m, f, e, and r are the numbers of male, females, environments, and replicates, respectively.The standard errors for GCA effects were calculated as described by Cox and Frey :SE(GCAf)Classifying newly developed early maturing QPM inbred lines into appropriate heterotic groups is important for the exploitation of maximum heterosis through crossing of inbreds from opposing heterotic groups . The 24 An inbred line was identified as a tester based on the following criteria proposed by Pswarayi and Vivek, : it shouijY is the average yield of genotype i in environment j, jY is the average yield across genotypes in environment j, i, j, i in environment j.A set of 22 QPM hybrids (top 12 and worst 10) including the four QPM hybrid checks were selected under CDHS and low-nitrogen conditions using the multiple trait selection base index values for the genotype main effect plus genotype by environment interaction (GGE) biplot analysis to separate the genotype by environment interactions into the component parts ,53. The P ≤ 0.05. The remaining secondary traits were regressed on the first order traits to identify those with significant contributions to grain yield and they were categorized as second order traits. The procedure was repeated in order to categorize the remaining traits into subsequent orders. The standardized b values generated by the step-wise regression analysis were the path coefficients [The step-wise multiple regression and sequential path diagrams were employed to determine the causal relationships between the measured traits under CDHS and low-nitrogen environments using the procedure described by Mohammadi et al. . The steficients ,55. The ficients  was doneThere was preponderance of additive gene action over non-additive one in the inheritance of grain yield under low-nitrogen and optimal environment conditions. The implication is that yield of maize hybrids under low-nitrogen and optimal environment conditions could be enhanced through recurrent selection and that inbred lines tolerant to low-nitrogen with high combining ability effects could be extracted from improved cycles of selection for hybrid development. Under CDHS environments, the non-additive gene action was more important than the additive gene action in the inheritance of grain yield implying that hybrid development should be employed under CDHS to exploit heterosis. Additionally, maternal effects influenced the inheritance of grain yield under CDHS environments. Plant aspect, ear aspect and ear height were identified as the primary traits contributing to the observed variations in grain yield under CDHS and low-nitrogen conditions. TZEQI 6 was identified as an inbred tester while hybrid TZEQI 113 × TZEQI 6 was identified as a single-cross tester. This implied that TZEQI 6 could be used either as a male or a female parent in the production of high yielding hybrids while hybrid TZEQI 113 × TZEQI 6 could be adopted as a single-cross tester for a three-way or double-cross hybrid production under low-nitrogen environments. Hybrids TZEQI 6 × TZEQI 228 and TZEQI 210 × TZEQI 188 should be tested extensively in on-farm trials and commercialized in SSA as combined low-nitrogen and CDHS tolerant hybrids.


The authors postulated that triceps deficiency can act as a surrogate marker of a more extensive plexus injury and may predict poorer outcomes if the weakness persists representing the trending differences between groups 2 and 3. However, given no statistical differences were demonstrated between groups 3 and 4, the authors conclude that utilising an abnormal triceps nerve that demonstrates sufficient strength and redundancy intraoperatively is preferable to alternative transfers for deltoid reanimation. Lastly, in group 4 patients where triceps nerves are damaged and unusable for nerve transfer, alternative operations can also achieve sufficient outcomes and should be considered for restoration of shoulder abduction.Upper brachial plexus injuries to the C5/6 roots or axillary nerve can result in severe deficits in upper limb function. Current techniques to reinnervate the deltoid muscle utilise the well-described transfer of radial nerve branches to triceps to the axillary nerve. However, in around 25% of patients, there is a failure of sufficient deltoid reinnervation. It is unclear in the literature if deltoid reanimation should be attempted with a nerve transfer from a weak but functioning triceps nerve. The authors present the largest series of triceps to axillary nerve transfers for deltoid reanimation in order to answer this clinical question. Seventy-seven consecutive patients of a single surgeon were stratified and analysed in four groups: (1) normal triceps at presentation, (2) abnormal triceps at presentation recovering to clinically normal function preoperatively, (3) abnormal triceps at presentation remaining abnormal preoperatively, and lastly (4) where pre-operative triceps function was deemed insufficient for use, requiring alternative reconstruction for deltoid reanimation. The authors considered deltoid re-animation of ≥ M4 as successful for the purpose of this study. Median Medical Research Council (MRC) values demonstrate group 1 achieves this successfully (M5), while median values for groups 2–4 result in M4 power . Median post-operative shoulder abduction active range of motion (AROM) values were represented by 170° (85–180) in group 1, 117.5° (97.5–140) in group 2, 90° (35–150) in group 3, and 60° (40–155) in group 4. For both post-operative assessments, subgroup analyses demonstrated statistically significant differences when comparing group 1 with groups 3 and 4 ( Injury to the upper brachial plexus (C5/C6 roots) or the axillary nerve results in devastating impairments of the function and aesthetics of the upper limb . The delLeechavengvongs et al. popularised the nerve transfer for re-animation of the injured deltoid muscle for shoulder abduction using the nerve to the long head of triceps to the anterior branch of the axillary nerve . This teThe transfer of radial nerve triceps branches to the axillary nerve is now well-described as a successful technique and the current preferred option of many brachial plexus surgeons for reinnervation of the deltoid. Bertelli et al. reported on 10 patients with C5/C6 root avulsions who underwent nerve to the long or lateral head of triceps to axillary nerve, SAN to SSN, and ulnar nerve fascicle to biceps motor branch transfers. All patients achieved active shoulder abduction in 2 years with three having Medical Research Council (MRC) muscle power scale scores of M4 (4/5) and seven scoring M3 , 11. MorDespite these reports of successful outcomes, Lee et al. observed that failure of meaningful deltoid reinnervation with this transfer reaches around 25% . FactorsAll nerve transfers require intact donor axons, with most stating a prerequisite donor strength of ≥ M4, yet this variable is not consistently reported , 10, 13.This study, therefore, aims to investigate this pre-operative surgical dilemma in order to inform clinical decision making. The surgical outcomes of a single surgeon performing triceps to axillary nerve transfers are evaluated and compared within three clinically distinct groups of patients:  normal tThe prospectively maintained database and clinical notes were examined for patients who had received nerve to triceps transfer for deltoid reanimation between 2007 and 2018 by a single surgeon working in private and public institutions in Melbourne, Australia. All the patients presented with brachial plexus palsies underwent surgical intervention for deltoid reanimation and had follow-up for an average of 33 months post-operatively.Pre-operative data collected included patient demographics, co-morbidities, injury demographics, level of brachial plexus injury, and pre-operative functional assessment of donor and recipient nerves. The patients were placed into groups according to their MRC score of triceps strength at both presentation and time of surgery  Table 1Table 1. Two independent reviewers collated all operative notes to record the procedure performed, intraoperative assessment of nerve branches to triceps, and details of nerve coaptations. These groups were segregated by documented pre-operative triceps function as described . By measuring the functional outcomes of deltoid reanimation, the authors aim to answer the clinical question at hand: can abnormal triceps nerves be used for axillary nerve transfer successfully?The patients were placed in four groups according to their presenting and pre-operative triceps assessment see .1) Normal triceps at presentation.2) Abnormal triceps at presentation recovering to clinically normal triceps function pre-operatively.3) Abnormal triceps at presentation remaining clinically abnormal at time of surgery.4) Pre-operative triceps function deemed insufficient for use, therefore, alternative reconstruction deltoid reinnervation.+/5 and is well-understood by clinicians as a real clinical subgroup and was described in an update in 1943 . This category is commonly referred to as a 4 in 1943 . This waThe operative procedure performed was similar to that previously described by Leechavengvongs et al. . The patIntraoperative stimulation offers different, and, in the practise of authors, much more useful information than pre-operative neurophysiological testing including EMG. The reason for this is because the essential questions in these patients become (a) are there enough sufficiently well-motored triceps nerves to use that will successfully reinnervate deltoid? and (b) if those nerves are sacrificed for the transfer, would there be sufficient residual triceps nerves for ongoing triceps function? This information is only possible to elicit by open dissection, then selective, insulated stimulation of each triceps nerve, which we routinely undertake at 0.5 mA. In this way, it is possible to accurately identify which triceps nerves are functioning and to what extent. This enables labelling those triceps nerves as either strongly functioning , weakly functioning (muscle seen to flicker but poor or no elbow extension) or non-functioning. Once this is complete, an assessment can be made as to whether there are sufficiently intact and strongly stimulating triceps nerves for both sacrifice to use in reconstruction while preserving good elbow extension. Generally, a minimum of three strongly stimulable triceps nerves  are preserved for triceps function.The procedure can safely be discontinued at this point if the evaluated donors demonstrate insufficient stimulation or insufficient redundancy to allow harvest while preserving adequately maintained triceps function. Once appropriate donors had been selected, donor neurotomies were undertaken as distally as required for tension-free transfers. The anterior axillary nerve was selectively the highest priority recipient nerve target. If motor components of intermediate or posterior axillary nerves were identified entering the deltoid muscle and sufficient donors were available, then further triceps to axillary nerve transfers were performed. Ultimately, it was most common for two or three  triceps nerves to be transferred in any individual case. Coaptation was performed under the microscope without tension using 9-0 monofilament nylon and fibrin glue applied to the outside of completed nerve repairs. We have a preference to use medial head triceps nerves for reconstruction and preserve lateral and long head if possible but ultimately will use and leave whichever nerves are required in an individual case. The reason the senior author generally preferred using medial head nerves is because of their typically favourable length, calibre, and axon counts as well as their more concealed donor site contour deformity.via a separate posterior shoulder approach during the same surgery. All the patients were placed in a shoulder immobiliser sling to protect nerve coaptations for 2 weeks. When reviewed at 2 weeks, the patients commenced mobilisation with the guidance of a hand therapist. Post-operative reviews were generally undertaken at ~3–6 monthly intervals thereafter.If a concurrent SAN to SSN transfer was planned, this was performed Post-operative outcomes included functional assessment comprising shoulder range of motion and MRC power grading. The time to plateau for a deltoid function was calculated as the time from surgery until the highest MRC shoulder abduction score or highest degree of shoulder abduction AROM was obtained and maintained at subsequent follow-up.Post-hoc pairwise comparisons were performed to determine differences among the four groups. A two-sided p < 0.05 was considered to be statistically significant. Analyses were performed with SAS software version 9.4 .Continuous variables were summarised using mean (standard deviation) or median (interquartile range) according to data type and distribution. Categorical variables were reported as counts and proportions. Comparisons between groups were made by the Krukal–Wallis test for continuous variables and chi-square or Fisher's exact test as appropriate for categorical variables. Between the years 2007 and 2018, 80 patients presented to the senior author (SF) for surgical management of traumatic brachial plexus injuries requiring deltoid reanimation. Of the 80 patients, three were excluded because of insufficient pre- and post-operative assessment documentation or loss to follow-up. Therefore, the authors present outcomes for a series of 77 patients. Sixty-four patients underwent nerve to triceps transfer for deltoid reanimation, further subcategorised according to their triceps MRC grading: 45 patients (group 1) with normal triceps preoperatively, 5 patients (group 2) with abnormal triceps that improved to a clinically normal triceps preoperatively, and 14 (group 3) with abnormal triceps that improved although remained clinically abnormal preoperatively. The triceps nerve donors in group 3 patients, although not normal, were deemed to be sufficient for nerve transfer during pre-operative assessment and intra-operative interrogation. Using the on-table risk minimisation strategy outlined in Methods, no patient suffered any problematic or concerning post-operative downgrading of triceps power. Most commonly when available, multiple medial head triceps nerves would be used for the reconstruction. One reason for this is the favourable length of such nerves, such that they can be transferred very close to the entry to the muscle of motor axillary nerves, thereby reducing time to reinnervation. Another reason is that frequently these young, body-conscious patients prefer, if possible, to preserve the visible contour of the more proximal triceps musculature, particularly lateral head.The remaining 13 patients (group 4) underwent alternative nerve transfers for deltoid reanimation as they had no suitable triceps nerve donors. These operations included radial nerve fascicular transfers, thoracodorsal nerve transfers, intercostal nerve transfers to axillary nerve, or nerve grafting from a C5 root . The breThe demographics of the 77 patients presented in this series demonstrate a mean age of 39.5 ± 4.4 years with a 90% male pre-dominance, with no significant differences between groups. The patterns of brachial plexus injury, as well as presentation and pre-operative triceps MRC grading, were significantly different and in keeping with the severity of the injury basis of group categorisation. The average time from injury to surgery was 6.5 ± 3.7 months across all groups with no significant difference between groups .p < 0.05). The authors appreciate MRC grading is a clinical measure reported in whole numbers but for purposes of analyses the above results are reported as both median values with the interquartile range (IR) and mean with standard deviation (STD) (p < 0.05) . Post-operative shoulder abduction MRC measurements are shown in on (STD) . All the < 0.05) . All othThe time to post-operative shoulder abduction MRC plateau  was not p < 0.05). Similar to MRC measurements, key findings on subgroup analyses demonstrated statistically significant differences when comparing group 1 with groups 3 and 4 (p < 0.05), while all the other group to group pairwise comparisons did not reach significance.Pre-operative shoulder abduction AROM demonstrated no significant difference between groups 1 to 4. Mean and median post-operative shoulder abduction AROM measurements are shown in p < 0.05) (The time to post-operative shoulder abduction AROM plateau was found to be significantly different when group 1 was compared with groups 3 and 4 ( < 0.05) .p < 0.05). In addition to these variables, the combined groups 1 and 2 patients demonstrated a significantly shorter time to AROM plateau , to patients who had abnormal pre-operative triceps (group 3) who underwent triceps to axillary nerve transfer. These results determined that the patients with normal pre-operative triceps function (combined groups 1 and 2) achieved improved post-operative shoulder abduction MRC and AROM scores compared with those in group 3 with abnormal triceps  resulted in a significant reduction in post-operative shoulder abduction MRC and AROM, and an increase in time to AROM plateau in comparison with group 1. The authors hypothesise that there is a reduced axonal count in groups 2 and 3 triceps nerve donors in comparison to the group 1 patients, which is represented by weakness in triceps function at presentation. While group 2 triceps function recovers, group 3 improves but continues to demonstrate abnormal triceps strength, which may account for slightly poorer reinnervation of the deltoid muscle target.The patients in group 4, with the unsuitable nerve to triceps donors, showed significantly lower post-operative shoulder abduction MRC and AROM scores, and an increase in time to AROM plateau in comparison with group 1. These patient groups in whom nerve to triceps was found to be abnormal or unusable (groups 3 and 4) were found to have a greater extent of injury to their brachial plexus with at least C5/6/7 involvement compared with C5/6 or isolated axillary nerve injuries (groups 1 and 2). The authors propose that due to additionally paralysed shoulder muscles , the more severely injured groups 3 and 4 patients will predictably have reduced shoulder functional outcomes, a finding that was supported by Rezzadeh et al. in a systematic review of SAN to SSN outcomes . This seThe authors have presented the largest series of triceps nerve to axillary nerve transfers for deltoid reanimation demonstrating successful results across all four sub-groups of patients. However, there are some limitations to this study. Despite the relatively large case series numbers in this field of surgery, the smaller number of patients in groups 2, 3, and 4 along with occasional missing data variables limited some of the statistical analyses . To presThe critical question the authors sought to answer is whether deltoid reanimation should be undertaken using functional but persistently abnormal nerves to triceps, and if so, under what circumstances are the triceps donor nerves still the best option for this group? These questions are addressed below with support from the results of this case series. The decision to proceed with the triceps to axillary nerve transfer in this series was based on a pre-operative triceps strength of ≥M4, as determined by the senior surgeon. This is a key determinant and a variable that is not often recorded or presented clearly in the literature. The senior author then conducted a thorough intraoperative assessment of all separate triceps nerves, which were required to display adequate strength in stimulation as well as redundancy before the decision to transfer was made.p < 0.05). Subgroup analysis of the patients in Group 1 who also underwent SAN to SSN transfer compared with those who underwent triceps to axillary nerve transfer alone did not demonstrate differences in post-operative shoulder abduction scores, demonstrating the efficacy of the results of nerve transfer in restoring all damaged components of shoulder abduction.The authors considered deltoid re-animation of ≥M4 as successful for the purpose of this study. Analyses using the median values  demonstrOn further subgroup examination, there were no differences in post-operative outcomes between groups 2 and 3 patients. The trends of improved post-operative MRC and AROM and a shorter AROM time to plateau in group 2 vs. 3 may be explained by improved axonal counts in group 2 where triceps nerve donors recover to normal prior to transfer, rather than persisting as abnormal. These findings are further supported by the subgroup analysis of patients with normal triceps in the pre-operative setting (groups 1 and 2 combined) vs. those with abnormal but usable triceps nerves (group 3), demonstrating improved post-operative shoulder abduction outcomes in the normal triceps groups, likely representing a less severe brachial plexus injury and improved axonal counts in the triceps nerves transferred for deltoid reanimation.Comparison of groups 3 and 4 patients did not demonstrate any significant differences in post-operative outcomes , 5. WhenIn conclusion, the current preference of the senior author is to utilise multiple triceps to axillary nerve transfers for restoration of deltoid function even if triceps nerves are observed to have less than normal function, as long as all criteria listed below are met:The triceps power by the time of surgery is clinically ≥M4.Surgical exploration confirms sufficient triceps donor nerve power.Surgical exploration reveals sufficient functioning triceps nerves for both powering the nerve transfer and maintaining adequate triceps strength.This approach is undertaken with the knowledge that while results for patients in groups 2 and 3 are inferior in post-operative outcomes in comparison to  group 1 patients, the observed outcomes are sufficiently successful that these transfers are equivalent or superior in most cases to alternative options used in group 4 patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by St Vincent's Private Hospital East Melbourne. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SF senior author and surgeon for all operative cases presented in this case series. LS and AW data collection, manuscript, and figure preparation. All authors contributed to the article and approved the submitted version.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.