CHO cell lines are a workhorse for the production of pharmaceutical proteins, but show some limitations in the variability and stability of N-glycosylation profiles. One promising approach to addressing this at the required systems-level is miRNA, which can regulate a large number of genes and have predictable targets. Herein, we first identified de novo 656 potential miRNAs in the CHO genome based on a combination of literature, database searching, and miRNA sequencing. We further sequenced mRNA from the same cultures, and used a combination of mRNA-miRNA correlation analysis, target prediction and literature searches to find miRNAs potentially targeting N-glycosylation. Our ten best miRNA candidates were subjected to miRNA overexpression, knockdown, or knock-out in CHO cell lines. Out of the ten candidates, four (miR-128, miR-34c, miR-30b, and miR-449a) showed positive effects on N-glycosylation and could be applied directly for CHO cell engineering. The fact that 40% of the screened targets had a desired effect, and the prediction of 656 miRNAs illustrates the massive potential of miRNA engineering in CHO.

The loss of genetic diversity is a challenge many species are facing, and genomics is a potential tool that can inform and prioritize decision making. Caribou populations have experienced significant recent declines throughout Québec, Canada, and some are considered threatened or endangered. We calculated the ancestral and contemporary patterns of genomic diversity of five caribou populations and applied a comparative framework to assess the interplay between demography and genomic diversity. We calculated a caribou specific mutation rate, μ, by extracting orthologous genes from related ungulates. Whole genome re-sequencing was completed on 67 caribou and genotype likelihoods were estimated. We calculated nucleotide diversity, θπ and estimated the coalescent or ancestral Ne, which ranged from 12,030 to 15,513. When compared to the census size, NC, the endangered Gaspésie Mountain caribou population had the highest Ne:NC ratio which is consistent with recent work suggesting high ancestral Ne:NC is of conservation concern. These ratios were highly correlated with genomic signatures (i.e. Tajima’s D) and explicit demographic model parameters. Values of contemporary Ne, estimated from linkage-disequilibrium, ranged from 11 to 162, with Gaspésie having among the highest contemporary Ne:NC ratio. Importantly, conservation genetics theory would predict this population to be of less concern based on this ratio. Of note, F varied only slightly between populations, and runs of homozygosity were not abundant in the genome. Our study highlights how genomic patterns are nuanced and misleading if viewed only through a contemporary lens; a holistic view should integrate ancestral Ne and Tajima’s D into conservation decisions.

Objectives: Women with Down syndrome (DS) suffer from several health issues, however, their fecundity is not affected. Despite that, there are no studies in the literature to address pregnancy, delivery, or neonatal outcomes among women with DS. Design: We conducted a retrospective study using the Health Care Cost and Utilization Project-Nationwide Inpatient Sample Database over 11 years from 2004 to 2014. Methods: A delivery cohort was created using ICD-9 codes. ICD-9 code 758.0 was used to extract the cases of maternal DS. Pregnant women with DS (study group) were matched based on age and health insurance type to women without DS (control) at a ratio of 1:4. A multivariant logistic regression model was used to adjust for statistically significant variables (P-value < 0.5). Results: There were a total of 9,096,788 deliveries during the study period. Of those, 185 pregnant women were found to have DS. The matched control group was 740. Maternal pregnancy risks mostly did not differ between those with and without DS including pregnancy-induced PIH, gestational diabetes, preeclampsia, PPROM, chorioamnionitis, cesarean section, operative vaginal delivery, or blood transfusion (P >0.05, all). However, they were at extremely increased risk of delivering prematurely (aOR 3.86, 95% CI 1.25-11.93), and to have adverse neonatal outcomes such as small for gestational age (aOR 13.13, 95% CI 2.20-78.41), intrauterine fetal demise (aOR 20.97, 95% CI 1.86-237.02), and congenital anomalies (aOR 9.59, 95% CI 1.47-62.72). Conclusion: Women with DS should be counseled about their increased risk of premature delivery and adverse neonatal outcomes.

Based on an equivalent derivative nonlinear Schr\”{o}inger equation, some periodic and non-periodic two-parameter solutions of the deformed continuous Heisenberg spin equation are obtained. These solutions are all proved to be ill-posed by the estimates of Fourier integral in ${H}^{s}_{\mathrm{S}^{2}}$ (periodic solution in ${H}^{s}_{\mathrm{S}^{2}}(\mathbb{T})$ and non-periodic solution in ${H}^{s}_{\mathrm{S}^{2}}(\mathbb{R})$ respectively). If $\alpha \neq 0$, the range of the weak ill-posedness index is $1

Pre-eclampsia screening studies - overcoming intervention biasHS CuckleFaculty of Medicine, Tel Aviv University, Ramat Aviv, IsraelThe ASPRE trial established beyond doubt the efficacy of aspirin prophylaxis in women with positive multi-marker first trimester preeclampsia (PE) screening test results (Rolnik et al. N Eng J Med 2017;50:613-22). Screening combined maternal characteristics, blood pressure, uterine artery Doppler, maternal serum pregnancy associated plasma protein (PAPP)-A and placental growth factor (PlGF). Screen-positive women were randomised to aspirin or placebo and there was a 62% reduction of pre-term PE in the aspirin arm.Subsequently, a practical question has arisen regarding the maternal serum markers: which is superior, PlGF or PAPP-A? This is best answered by non-intervention studies of PE screening when all markers are measured prospectively. There are four such studies and all show that the detection rate for a fixed 10% false-positive rate was higher when PlGF was included compared with PAPP-A; the increase ranged from 5% to 7% (Cuckle. Ultrasound Obstet Gynecol 2022;??:??-??).However, a non-intervention study, despite not revealing the PE screening test report to clinicians and patients, does not preclude the use of aspirin in some women; for example, those with high risk characteristics are likely to be recommended treatment. Moreover, these occasional interventions might bias the PlGF versus PAPP-A comparison. This would occur as a consequence of simultaneous Down syndrome screening using the Combined Test, since that test report includes the PAPP-A level. If this marker was low, treatment might be recommended, leading to the prevention of some pre-term PE cases, a proportion of which are screen-positive. In the absence of intervention these screen-positive cases would be true-positive but actually become false-positive, hence reducing the detection-rate and slightly increasing the false-positive rate. These effects will be stronger for PAPP-A combinations. The standard Combined Test does not include PlGF yielding a bias towards superior PE screening performance for PlGF combinations.In the current analysis, data from two of the four non-intervention studies are reanalysed to adjust for this potential bias (Wright et al. BJOG 2022;??:??-??). In both combined 4.2% (1066/25,226) had taken aspirin, although for nearly all the treatment was sub-optimal compared with the ASPRE regimen of 150mg/night at <16-36 weeks. The reanalysis was by statistical modeling using the original ‘competing risks’ method. But additionally superimposed were simulations from an ‘imputation’ model, which re-assigned false-positives among the 1066 treated women to true- or false-positive according to the probability of reduction in pre-term PE found in ASPRE.The model predicted that the increase in detection rate for a 10% false-positive rate when PlGF was included compared with PAPP-A was 7.0% and this reduced to 6.4% following imputation. Hence, even adopting the extreme assumption that intervention was at an optimal level, the bias in favour of PlGF was small. The authors also modeled combinations without blood pressure or uterine artery Doppler and the bias was proportionally similar or smaller.Clinical studies are often marred by subtle bias, and once discovered it is vital to assess whether the results were materially affected. The current publication is exemplary in using imputation modeling to confirm the superiority of PlGF over PAPP-A.(500 words)

This paper investigates the boundary finite-time stabilization of fractional reaction-diffusion systems (FRDSs). First, a distributed controller is designed, and sufficient conditions are obtained to ensure the finite-time stability (FTS) of FRDSs under the designed controller. Then, a boundary controller is presented to achieve the FTS. By virtue of Lyapunov functional method and inequality techniques, sufficient conditions are presented to ensure the FTS of FRDSs via the designed boundary controller. The effect of diffusion term of FRDSs on the FTS is also investigated. Both Neumann and mixed boundary conditions are considered. Moreover, the robust finite-time stabilization of uncertain FRDSs is studied when there are uncertainties in the system’s coefficients. Under the designed boundary controller, sufficient conditions are presented to guarantee the robust FTS of uncertain FRDSs. Finally, numerical examples are presented to verify the effectiveness of our theoretical results.

Consumers are becoming aware of the relevance of eating low levels of trans and saturated fats in processed foods. In addition, many countries are adopting regulatory measures on the use of these ingredients. For this reason, the exploration of new technologies capable of producing structures that trap liquid oil (composed of unsaturated fatty acids, considered healthier) has been widely investigated in order to replace saturated and trans fats in food products. One of the most promising technologies is the so-called oleogels, which present a great challenge to mimic sensory attributes related to the texture of processed foods based on saturated fats. In this review, we discuss how the different approaches used in the production of oleogels, direct or indirect methods, as well as compositional variables, such as oleogelators and mixing ratio, can directly influence the mechanical properties of these structures. An overview of the parameters that can interfere with these properties contributes to a better understanding of the building of the oleogels and their possible applications.

The Geometric Algebra formalism opens the door to developing a theory upgrading conventional quantum mechanics. Generalizations, stemming from implementation of complex numbers as geometrically feasible objects in three dimensions; unambiguous definition of states, observables, measurements bring into reality clear explanations of conventional weird quantum mechanical features, particularly the results of double split experiments where particles create diffraction patterns inherent to wave diffraction. This weirdness of the double split experiment is milestone of all further difficulties in interpretation of quantum mechanics.

For over a decade, experimental evolution has been combined with high-throughput sequencing techniques in so-called Evolve-and-Resequence (E&R) experiments. This allows testing for selection in populations kept in the laboratory under given experimental conditions. However, identifying signatures of adaptation in E&R datasets is far from trivial, and it is still necessary to develop more efficient and statistically sound methods for detecting selection in genome-wide data. Here, we present Bait-ER - a fully Bayesian approach based on the Moran model of allele evolution to estimate selection coefficients from E&R experiments. The model has overlapping generations, a feature that describes several experimental designs found in the literature. We tested our method under several different demographic and experimental conditions to assess its accuracy and precision, and it performs well in most scenarios. However, some care must be taken when analysing specific allele trajectories, particularly those where drift largely dominates and starting frequencies are low. We compare our method with other available software and report that ours has generally high accuracy even for very difficult trajectories. Furthermore, our approach avoids the computational burden of simulating an empirical null distribution, outperforming available software in terms of computational time and facilitating its use on genome-wide data. We implemented and released our method in a new open-source software package that can be accessed at https://github.com/mrborges23/Bait-ER.

This article proposes an adaptive neural output feedback control scheme in combination with state and disturbance observers for uncertain fractional-order nonlinear systems containing unknown external disturbance, input saturation and immeasurable state. The radial basis function neural network (RBFNN) approximation is used to estimate unknown nonlinear function, and a state observer as well as a fractional-order disturbance observer is developed simultaneously by using the approximation output of the RBFNN to estimate immeasurable states and unknown compounded disturbances, respectively. Then, a fractional-order auxiliary system is constructed to compensate the effects caused by the saturated input. In addition, by introducing a dynamic surface control strategy, the tedious analytic computation of time derivatives of virtual control laws in the conventional backstepping method is avoided. The proposed method guarantees that the boundness of all signals in the closed loop system and the tracking errors converge to a small neighbourhood around the origin. Finally, two examples are provided to verify the effectiveness of the proposed control method.

Silicon-based anodes with lithium ions as charge carriers have the highest predicted theoretical specific capacity of 3579 mA h g-1 (for Li15Si4). Contemporary electrodes do not achieve this theoretical value largely because conventional production paradigms rely on the mixing of weakly coordinated components. In this paper, a semi-conductive triazine-based graphdiyne polymer network is grown around silicon nanoparticles directly on the current collector, a copper sheet. The porous, semi-conducting organic framework (i) adheres to the current collector on which it grows via cooperative van der Waals interactions, (ii) acts effectively as conductor for electrical charges and binder of silicon nanoparticles via conjugated, covalent bonds, and (iii) enables selective transport of electrolyte and Li-ions through pores of defined size. The resulting anode shows extraordinarily high capacity at the theoretical limit of fully lithiated silicon. Finally, we combine our anodes in proof-of-concept battery assemblies using a conventional layered Ni-rich oxide cathode.

Confluent and reticulated papillomatosis (CRP) is a rare skin condition of uncertain etiology. The anti-bacterial and most importantly the anti-inflammatory mechanisms of some antibiotics seem to explain the effectiveness of these medications. Other measures such as reducing weight and treating an underlying endocrine disorder may be helpful.

IntroductionSinus venosus atrial septal defects (SVASDs), originally described in 1858, account for approximately 4%-11% of all atrial septal defects (ASDs)[1,2]. The typical SVASD is an interatrial communication that results from a deficiency of the common wall between the superior vena cava (SVC) and the right-sided pulmonary veins[2,3]. SVASD is commonly associated with anomalous pulmonary venous connections (APVCs) involving some or all of the pulmonary veins[3,4], which produces additional left-to-right shunting. The basic principle of repair is redirection of the APVC through the interatrial communication into the left atrium.In contrast to operative repair of secundum ASD, the surgical approach for SVASD is more complex and carries the risk of stenosis of the SVC or pulmonary veins, residual shunting, and sinus node dysfunction[4].We reviewed outcomes for patients who underwent SVASD repair at Bhanubhai and Madhuben Patel Cardiac Centre, Karamsad, Anand, Gujarat, India, with a focus on patient survival and development of arrythmias.

Background The use of thoracic endovascular aortic repair (TEVAR) for the management of uncomplicated type B aortic dissection (un-TBAD) remains controversial. There is a lack of consensus over whether pre-emptive TEVAR should be carried out in patients with un-TBAD at risk of progression to complicated TBAD. We present a review of current evidence and seek to suggest criteria where endovascular intervention in un-TBAD may prove beneficial relative to pharmacotherapy alone. Methods and Materials PubMed and Cochrane databases were searched using terms including: type B aortic dissection, risk factors, medical therapy, TEVAR, false lumen expansion, and mortality. Papers were selected based on title and abstract. Results Optimal medical therapy remains the mainstay treatment for patients with un-TBAD, however patients with un-TBAD present with varying degrees of disease progression risk. Factors such as age, aortic morphology, history of connective tissue disorders, false lumen thrombosis, and aortic branch involvement may potentiate progression from un-TBAD to complicated TBAD. Short- and long-term outcomes associated with TEVAR for TBAD remain promising. Conclusion Pre-emptive TEVAR may be beneficial in patients with un-TBAD presenting with the above factors, however further prospective research into the optimal timing for TEVAR in un-TBAD is required.

Bilateral lung transplant for pulmonary hypertension with pulmonary artery aneurysmBerhane Worku MD1,2, Charles Mack MD1,3, Ivancarmine Gambardella MD1,2New York Presbyterian Weill Cornell Medical Center, New York NY 10021New York Presbyterian Brooklyn Methodist Hospital, Brooklyn NY 11215New York Presbyterian Queens Hospital, Queens NY 11335Corresponding AuthorBerhane Worku MD Brooklyn Methodist HospitalDepartment of Cardiothoracic Surgery506 6th StreetBrooklyn, NY 11215718-780-7700Bmw2002@med.cornell.eduPulmonary artery aneurysms (PAA) may be secondary to congenital cardiac defects such as a patent ductus arteriousus (PDA), atrial septal defect, or ventricular septal defect. They may also occur secondary to infection or connective tissue disease or they may be idiopathic in nature. Repair is undertaken to prevent the sequelae of rupture or dissection, although the specific size criteria at which repair is recommended remains controversial. Pulmonary hypertension (PH) may also lead to PAA, in which case isolated repair is not recommended. Heart-lung transplant has classically been the treatment of choice for PH with PAA, especially when associated with congenital heart defects, right ventricular dysfunction, and pulmonic valve regurgitation.In the setting of PH with PAA and correctable cardiac defects, bilateral lung transplant (BLT) has been described. Concurrent PAA repair is required, and several techniques exist to allow for this. In the current issue of the Journal of Cardiac Surgery, Doi et. al. offer a review of PH with PAA, with a focus on strategies to allow for BLT and PAA repair, hence avoiding the need for HLT. They describe a case of a patient with PH secondary to a PDA and a 9cm PAA who underwent BLT and PAA repair. The donor descending aorta and a bovine pericardial tube was used to reconstruct the recipient main and right PA, respectively. The patient suffered from persistently elevated PA pressures postoperatively due to a kink in the anastomosis between the neo-main PA (donor descending aorta) and the neo-right PA (bovine pericardial tube) requiring surgical revision, but the patient otherwise made an excellent recovery1.The benefit with BLT (rather than HLT) stems from limitations in donor supply which may result in unacceptably long wait times and reduced waitlist survival in patients awaiting HLT. As right ventricular function typically improves after BLT for PH, the donor heart from a HLT bloc may be better served to another patient with terminal cardiac failure. A variety of techniques have been described to allow for repair of massive PAAs at the time of BLT. Harvesting of the entire donor PA to allow replacement of the PAA has been described and is feasible when the donor heart is unsuitable for transplantation2,3. When the donor main PA is unavailable for harvesting, pulmonary arterioplasty and replacement with donor descending aorta have been described at the time of BLT4-7. After resection of the PAA, the proximal donor aorta is anastamosed to the proximal PA with the distal aorta oriented towards the right lung. The distal donor aorta is anastamosed to the donor right PA. The innominate and left carotid orifices can be used for anastomoses to the donor left PA5,6. Extension of a short donor left PA with an autologous pericardial tube has been described5. Similarly, extension of a short donor right PA with a bovine pericardial tube is described in the current report1.Pulmonary valve (PV) regurgitation may occur secondary to annular dilation from the PAA. PV replacement has been described, including sutureless valve implantation with valves intended for percutaneous deployment4. Durability remains a concern, and valve sparing repair techniques (commisuroplasty) have also been described3. When the donor heart is not being harvested, BLT with procurement of the donor right ventricular outflow graft has been described8. HLT always remains a reasonable option in the setting of extremely massive PAA associated with severe PV regurgitation and right ventricular dysfunction, assuming adequate donor availability and ability of the recipient to tolerate the longer wait time9.Recovery of right ventricular function and tricuspid regurgitation after BLT for PH has been documented, supporting the shift from HLT to BLT for this entity. In the setting of left ventricular diastolic dysfunction, severe pulmonary edema and hypoxia can be seen after BLT for PH as the LV is suddenly loaded, and in such a scenario ECMO has been utilized to allow time for LV remodeling. Various centers may prefer HLT over BLT for these cardiac consequences of prolonged PH10. In the absence of these complicating factors, BLT should be considered for PH in otherwise appropriate candidates. BLT for PH with PAA is likely best managed with harvesting the donor main PA when the donor heart is not being considered for harvest. When the donor PA is not available, the decision to attempt the abovementioned strategies for PAA repair such as neo-PA creation with donor aorta and the associated prolongation of donor ischemic time must be weighed against exposing the patient to elevated waitlist mortality while waiting for an acceptable heart-lung bloc to become available. Transplant center expertise and regional differences in heart and lung donor utilization rates will likely a relevant factor to consider when selecting the optimal strategy for each patient.REFERENCESDoi A, Gajera J, Niewodowski D, Gangahanumaiah S, Whitford H, Snell G, Kaye D, Joseph T, McGriffin D. Surgical management of giant pulmonary artery aneurysms in patients with severe pulmonary arterial hypertension. J Card Surg; in press]Schwarz S, Benazzo A, Prosch H, Jaksch P, Klepetko W, Hoetzenecker K. Lung transplantation for pulmonary hypertension with giant pulmonary artery aneurysm. J Thorac Cardiovasc Surg 2020;159:2543-50Shayan H, Sareyyupoglu B, Shigemura N, Thacker J, Bermudez C, Toyoda Y. Lung transplant, double valve repair, and pulmonary artery aneurysm resection. Ann Thorac Surg 2012;93:e3-5Pelenghi S, Primiceri C, Belliato M, Ghio S, Scelsi L, Totaro P. Is it time for a paradigm shift: Should double-lung transplant be considered the treatment of choice for idiopathic pulmonary arterial hypertension and giant pulmonary aneurysm? J Card Surg 2021;36:2996-2999Noda M, Okada Y, Saiki Y, Sado T, Hoshikawa Y, Endo C, Sakurada A, Maeda S, Oishi H, Kondo T. Reconstruction of pulmonary artery with donor aorta and autopericardium in lung transplantation. Ann Thorac Surg 2013;96:e17-9Force SD, Lau CL, Moazami N, Trulock EP, Patterson GA. Bilateral lung transplantation and pulmonary artery reconstruction in a patient with chronic obstructive pulmonary disease and a giant pulmonary artery aneurysm. J Thorac Cardiovasc Surg 2003;126:864-6.Oda H, Hamaji M, Motoyama H, Ikeda T, Minatoya K, Nakajima D, Chen-Yoshikawa TF, Date H. Use of a three-dimensional model in lung transplantation for a patient with giant pulmonary aneurysm. Ann Thorac Surg 2020;109:e183-5Zanotti G, Hartwig MG, Davis RD. A simplified technique for pulmonary artery aneurysm repair in a lung transplant recipient with right ventricular outflow tract obstruction. J Thorac Cardiovasc Surg 2013;145: 295-6Eadington T, Santhanakrishnan K, Venkateswaran. Heart-lung transplantation for idiopathic pulmonary arterial hypertension and giant pulmonary artery aneurysm – case report. J Cardiothorac Surg 2020;15:169Budev MM, Yun JJ. Advanced circulatory support and lung transplantation in pulmonary hypertension.

A prominent problem in computer vision is occlusion, which occurs when an object’s key features temporarily disappear behind another crossing body, causing the computer to struggle with image detection. While the human brain is capable of compensating for the invisible parts of the blocked object, computers lack such scene interpretation skills. Cloud computing using convolutional neural networks is typically the method of choice for handling such a scenario. However, for mobile applications where energy consumption and computational costs are critical, cloud computing should be minimized. In this regard, we propose a computer vision sensor capable of efficiently detecting and tracking covered objects without heavy reliance on occlusion handling software. Our edge-computing sensor accomplishes this task by self-learning the object prior to the moment of occlusion and uses this information to “reconstruct” the blocked invisible features. Furthermore, the sensor is capable of tracking a moving object by predicting the path it will most likely take while travelling out of sight behind an obstructing body. Finally, sensor operation is demonstrated by exposing the device to various simulated occlusion events. Keywords:  Computer vision, occlusion handling, edge computing, object tracking, dye sensitized solar cell. Corresponding author Email: hoinok.tsao@nie.edu.sg  

For more than three decades, the Asian turtle crisis has resulted in the decline of every native species in China. For some species, such as the yellow pond turtle (Mauremys mutica), wild populations have dwindled to near functional extinction. Previous studies show there is deep genetic divergence of M. mutica sensu lato between populations north and south of the Pearl River Drainage but no data to show if phylogeographic structure occurs within these two main types. In this study, we found clear phylogeographic structure. In northern types, we found two main clades, corresponding to mainland China and island clades (Taiwan and Yaeyema Islands) with uncorrected p values of 0.00-2.0% divergence in our 2353 bp concatenated mtDNA data set. For the southern types, we found three main clades corresponding to Hainan, Mainland (Vietnam/Guangxi) and the Annam pond turtle (Mauremys annamensis) with divergence ranging from 1.0-1.8% among these three groups. Moreover, the identification of northern and southern types by phenotype was roughly 98% accurate, which, coupling with the deep genetic divergence in mtDNA (5.5-6.7%) and in the 6056 bp nuDNA data set (0.16-0.37%) provide sufficient evidence for northern M. mutica to be an independent species, and individuals from the southern clade should be regarded as subspecies of M. annamensis. Finally, we provide the most comprehensive database to date which can be used to determine the region of origin for captive stock. Making the large captive populations of M. mutica, under the right conditions, potentially valuable for restocking or augmentation of wild populations.

Letter to the Editor

Global change is altering patterns of community assembly, with net outcomes dependent on species’ responses to the environment, both directly and mediated through biotic interactions. Here, we assess alpine plant community responses in a 15-year factorial nitrogen addition, warming and snow manipulation experiment. We used a dynamic competition model to estimate the density-dependent and independent processes underlying changes in species-group abundances over time. Density-dependent shifts in competitive interactions drove long-term changes in abundance of species-groups under global change. Density-independent processes were important when counteracting environmental drivers limited the growth response of the dominant species. Furthermore, competitive interactions shifted with environmental change, primarily with nitrogen, and drove non-linear abundance responses across environmental gradients. Our results highlight that global change can either reshuffle species hierarchies or further favor already dominant species; predicting which outcome will occur requires incorporating both density-dependent and independent mechanisms and how they interact across multiple global change factors.

Root knot nematode (RKN), a kind of plant parasitic nematodes, leads to large reduction of crop yield, and seriously damages the agricultural production. The phytohormone jasmonates (JAs) act as important signals to regulate resistance against multiple abiotic and biotic stresses. However, little is known about the mechanism of JA-mediated defense responses against RKN in tomato. In this study, we found that the WRKY transcription factor SlWRKY45 interacts with most of the Jasmonate-ZIM domain proteins (JAZs) in yeast and plant. Overexpression of SlWRKY45 decreased plant resistance to RKN Meloidogyne incognita with increased gall index. We further generated slwrky45 mutants using the CRISPR/Cas9 technology, and discovered that the gall index and the number of nematodes and females in slwrky45 mutants are significantly reduced compared with wild type, as inoculated with RKN Meloidogyne incognita. Moreover, the contents of jasmonic acid and JA-isoleucine (JA-Ile) were highly increased in slwrky45 mutants with RKN Meloidogyne incognita infection compared with wild type. Furthermore, EMSA, and Dual-LUC assays demonstrated that SlWRKY45 directly binds and represses jasmonate biosynthesis gene ALLENE OXIDE CYCLASE ( AOC). Overall, our findings reveled that JAZ-interaction protein SlWRKY45 negatively controls plant defense against RKN Meloidogyne incognita by the regulation of JA biosynthesis in tomato.

Plants in suburban forests of eastern North America face the dual stressors of high white-tailed deer density and invasion by nonindigenous plants. The combination of chronic deer herbivory and strong competition from invasive plants could alter a plant’s stress- and defense-related secondary chemistry, especially for long-lived juvenile trees in the understory, but this has not been studied. We measured foliar total antioxidants, phenolics, and flavonoids in juveniles of two native trees, Fraxinus pennsylvanica (green ash) and Fagus grandifolia (American beech), growing in six forests in the suburban landscape of central New Jersey, USA. The trees grew in experimental plots that had been subject for 2.5 years to factorial treatments of deer access/exclosure X addition/no addition of the nonindigenous invasive grass Microstegium vimineum (Japanese stiltgrass). As other hypothesized drivers of plant secondary chemistry, we also measured non-stiltgrass herb layer cover, light levels, and water availability. Univariate mixed model analysis of the deer and stiltgrass effects and multivariate structural equation modeling (SEM) of all variables showed that both greater stiltgrass cover and greater deer pressure induced antioxidants, phenolics, and flavonoids, with some variation between species. Deer were generally the stronger factor, and stiltgrass effects were most apparent at high stiltgrass density. SEM also revealed that soil dryness directly increased the chemicals; deer had additional positive, but indirect, effects via influence on the soil; in beech PAR positively affected flavonoids; and herb layer cover had no effect. Juvenile trees’ chemical defense/stress responses to deer and invasive plants can be protective, but also could have a physiological cost, with negative consequences for recruitment to the canopy. Ecological implications for species and their communities will depend on costs and benefits of stress/defense chemistry in the specific environmental context, particularly with respect to invasive plant competitiveness, extent of invasion, local deer density, and deer browse preferences.

Pilot Study of Nuclear Scintigraphy To Assess Cough Clearance in DMDTo the Editor:Respiratory muscle weakness as a consequence of neuromuscular disease results in ineffective cough, atelectasis, and pneumonia. Augmented airway clearance treatments such as mechanical in-exsufflation and high frequency chest compression have been shown to decrease morbidity and mortality in children with neuromuscular disease, although the evidence supporting these therapies in clearance of airway secretions is indirect.We sought to pilot the use of nuclear imaging of the clearance of pulmonary secretions before and after voluntary coughing and airway clearance treatment to explore the role of measures of pulmonary function to predict airway clearance. This technique has been used for some time in patients with cystic fibrosis but has not been applied to patients with neuromuscular disease or to assess the utility of airway clearance devices. To this end, we recruited 7 outpatient subjects (7.8-21 years of age) with Duchenne Muscular Dystrophy when clinically well and categorized as “Early Ambulatory” (requiring minimal assistive devices, n=4); “Early Non-ambulatory” (using wheelchair some of time, n=2); and “Late Non-Ambulatory” (always using wheelchair, n=1). This study was approved by local Institutional Review Board.Spirometry was performed according to ATS specifications and normalized using Global Lung Initiative equations. Peak cough flow was measured from total lung capacity (TLC) and data was normalized with previously published equations[1]. Maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) were measured at the mouth using a hand-held pressure manometer and maximal pressure from FRC was also be measured at the nares (SNIP) during a sniff maneuver[2] using a small nasal occluder and handheld transducer Maximal static pressures and SNIP pressures were normalized using previously published data.For imaging of mucus clearance, 4 mCi of Technetium [Tc-99m] sulfur-colloid particles in 2 ml of normal saline were delivered by nebulizer using a defined breathing pattern for 2 minutes [3]. During continuous imaging, subjects were asked to voluntarily cough every 10 seconds for 1 minute using guidance from a metronome, and this sequence was repeated 3 times. The subject then received an airway clearance treatment for 30 minutes using a high frequency chest compression device with pressure adjusted between 80-100% of maximum, and compression frequencies cycled between 5Hz-15Hz. The device was paused every 10 minutes at which time the patient was asked to cough every 10 seconds over 1 minute.Image analysis was performed according to previously published methods. Measurements of radioactivity in the right lung were corrected for background and radioactive decay and normalized by starting radioactive counts. A single retention curve was generated from the initial cough clearance period and the period during which the high frequency chest compression device was operated. Cough clearance (CC) represents the percent of total deposited radioactivity cleared during the associated period. We designated the percentage cleared by 18 voluntary coughs over 3 minutes as CCvol, and the percentage cleared during three 10 min periods of HFCC and 18 voluntary coughs over 3 minutes as CCHFCC. CCvol and CCHFCCwere correlated with physiologic measures including peak cough flow (PCF), MIP/MEP, and SNIP using Spearman’s correlation.Pulmonary function was overall well preserved (FVC 92% ± 35%). Respiratory muscle strength (in cmH2O) decreased with disease severity (e.g. MIP EA 73.5 ± 26, ENA 62.5 ± 14.8, LNA 47). Peak cough flow (% predicted) decreased with severity (EA 94±11, ENA 79±9, LNA 46). Complete details are provided in the online supplement.Representative imaging of two subjects is shown (Figure 1). Significant intra-subject differences were seen in clearance with voluntary cough and with cough augmented with HFCC vest (Figure 2). Voluntary cough was responsible for 46-96% of the total clearance and HFCC-augmented clearance for 3-53% of total clearance. For most patients, the kinetics of clearance with HFCC-augmented cough did not appear very different than for voluntary cough.Statistical comparisons between disease stages were hampered by the small sample size in each group. Nonetheless, while there appeared to be a trend for decreasing CCvol with stage of disease (Figure 2), age, and PCF, we found no statistically significant correlations between voluntary cough clearance and measures of pulmonary function (FVC, r=0.39), respiratory muscle strength (PCF, r=-0.63; SNIP r=-0.12, MIP r=0.43, MEP r=0.29), age (r=-0.57), height (r=-0.49), weight (r=-0.37). Additionally, we did not find statistically significant correlations between augmented cough clearance and measures of pulmonary function (FVC, r=0.40), respiratory muscle strength (PCF, r=-0.08; SNIP r=-.02, MIP r=-0.07, MEP r=0.05), age (r=-0.39), height (r=-0.75), weight (r=-0.03).This study is the first we are aware of to directly examine cough clearance of secretions using nuclear scintigraphy in patients with DMD. Importantly, the measurements were well tolerated by subjects. We did not find a relationship between CPF, respiratory muscle strength, or spirometry and voluntary cough clearance. However, these results must be qualified based on the small number of enrolled subjects. We do note that many of the patients had good cough clearance. Additionally, HFCC did not consistently alter the rate of secretion clearance above noted with voluntary cough.While Bach et al[4] showed that PCF >160 L/min was a predictor of successful tracheal decannulation in adults with neuromuscular disease, Airen et al[5] found that the majority of healthy children <13 years old generated PCFs < 270 l/min despite having the ability to generate MEP > 60 cmH2O. This suggests that PCF may change with age and that an absolute threshold cannot be used across the age span. There are no studies specifically examining use of HFCC in patients with DMD. As patients with DMD are expected to have impairment in cough, mechanical in-exsufflation would be expected to assist with airway clearance by replacing the function of weak respiratory muscles in a way that HFCC does not. Lechtzin et al[6] showed that in adults with neuromuscular diseases, HFCC decreased inpatient costs and costs for treating pneumonia compared to the year prior to initiating HFCC. Measures of pulmonary function, including PCF, were not assessed. It would be of interest to examine secretion removal with in-exsufflation using this nuclear imaging method.While we were not able to demonstrate a relationship between cough clearance and measures of respiratory muscle strength, peak cough flow, or FVC, it is possible that this relationship exists and would be demonstrated in a larger population. However, the longstanding assumption that PCF is a reliable indicator of need for airway clearance, especially in children, is not supported by these data. The biggest limitation of our pilot study is the small sample size. We suspect enrollment was limited due to preference for interventional studies. Another limitation is that for our population, the impairment of respiratory muscle strength was mild for most subjects. Additional subjects with more advanced weakness may have helped clarify the relationship between respiratory muscle strength and airway clearance. Finally, there can be day-to-day variations of clearance.There are now a number of airway clearance technologies being proposed for patients with neuromuscular and airway diseases (e.g. cystic fibrosis, ciliary dyskinesia). These include high frequency chest compression vests, battery-powered vests, in-exsufflation devices (with or without oscillation), intrapulmonary percussive ventilation devices, and handheld oscillating positive expiratory pressure devices. We propose that the imaging methodology used in this study could be utilized to compare these different techniques, some of which are costly and with limited or no comparison data. In addition, there are patients that will be unable to cooperate with pulmonary function measurements and an imaging assessment of airway clearance may be useful to determine optimal strategies.In conclusion, we demonstrated that use of nuclear scintigraphy is a feasible technique to examine airway clearance for patients with respiratory muscle weakness, as it has been used for patients with cystic fibrosis. Additional studies will be needed to further examine the relationship between pulmonary function measures and secretion removal.Daniel J. Weiner MD1,Hoda Abdel Hamid MD2,Timothy E. Corcoran PhD3Divisions of Pediatric Pulmonology1 and Neurology2, Department of PediatricsUPMC Children’s Hospital of PittsburghDivision of Pulmonary, Allergy and Critical Care Medicine3, Department of MedicineUniversity of Pittsburgh School of Medicine

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