The AtERF19 gene regulates meristem activity and flower organ size in plants.

Ethylene-responsive factors (ERFs) have diverse functions in the regulation of various plant developmental processes. Here, we demonstrate the dual role of an Arabidopsis ERF gene, AtERF19, in regulating reproductive meristem activity and flower organ size through the regulation of genes involved in CLAVATA-WUSCHEL (CLV-WUS) and auxin signaling, respectively. We found that AtERF19 stimulated the formation of flower primordia and controlled the number of flowers produced by activating WUS and was negatively regulated by CLV3. 35S::AtERF19 expression resulted in significantly more flowers, whereas 35S::AtERF19 + SRDX dominant-negative mutants produced fewer flowers. In addition, AtERF19 also functioned to control flower organ size by promoting the division/expansion of the cells through activating Small Auxin Up RNA Gene 32 (SAUR32), which positively regulated MYB21/24 in the auxin signaling pathway. 35S::AtERF19 and 35S::SAUR32 resulted in similarly larger flowers, whereas 35S::AtERF19 + SRDX and 35S::SAUR32-RNAi mutants produced smaller flowers than the wild type. The functions of AtERF19 were confirmed by the production of similarly more and larger flowers in 35S::AtERF19 transgenic tobacco (Nicotiana benthamiana) and in transgenic Arabidopsis which ectopically expressed the orchid gene (Nicotiana benthamiana) PaERF19 than in wild-type plants. The finding that AtERF19 regulates genes involved in both CLV-WUS and auxin signaling during flower development significantly expands the current knowledge of the multifunctional evolution of ERF genes in plants. The results presented in this work indicate a dual role for the transcription factor AtERF19 in controlling the number of flowers produced and flower organ size through the regulation of genes involved in CLV-WUS and auxin signaling, respectively. Our findings expand the knowledge of the roles of ERF genes in the regulation of reproductive development.

Single-cell RNA sequencing of solid pseudopapillary neoplasms of the pancreas in children.

Solid pseudopapillary neoplasm (SPN) of the pancreas is a rare pancreatic tumor in children. Its origin remains elusive, along with its pathogenesis. Heterogeneity within SPN has not been previously described. In addition, low malignant but recurrent cases have occasionally been reported. To comprehensively unravel these profiles, single-cell RNA sequencing was performed using surgical specimens. We identified the cell types and suggested the origin of pancreatic endocrine progenitors. The Wnt/ß-catenin pathway may be involved in tumorigenesis, while the epithelial-to-mesenchymal transition may be responsible for SPN recurrence. Furthermore, NOV, DCN were nominated as primary and S100A10, MGP as recurrent SPN marker genes, respectively. Our results provide insight into the pathogenesis of SPN.

Divergent Synthesis of Highly Substituted Tetrahydroquinolines and Cyclopentenes via Lewis Base Catalyzed Switchable [4 + 2] and [3 + 2] Annulations of MBH-Carbonates with Activated Olefins.

A highly selective and divergent synthesis which enabled access to various complex compounds is highly attractive in organic synthesis and medicinal chemistry. Herein, we developed an effective method for divergent synthesis of highly substituted tetrahydroquinolines via Lewis base catalyzed switchable annulations of Morita-Baylis-Hillman carbonates with activated olefins. The reaction displayed switchable [4 + 2] or [3 + 2] annulations via catalyst or substrate control, providing a diverse range of architectures which contained highly substituted tetrahydroquinolines or cyclopentenes with three contiguous stereocenters bearing a quaternary carbon center in high yields with excellent diastereoselectivities and regioselectivities. Furthermore, synthetic utility of this strategy was further highlighted by gram-scale experiments and simple transformations of the products.

Anemia Is Associated with Disease Severity, Hepatic Complications, and Progression of Wilson Disease: A Retrospective Cohort Study.

INTRODUCTION: Anemia is a common manifestation of chronic liver diseases. It is a predictor of severe disease, a high risk of complications, and poor outcomes in various liver diseases. However, it remains unclear whether anemia serves as a similar indicator in patients with Wilson disease (WD). Therefore, this study aimed to investigate the relationship between anemia and severity, hepatic complications, and the progression of WD. METHODS: Medical data were collected retrospectively from January 1, 2016, to December 31, 2020. Univariate and multivariate analyses were carried out to investigate the relationship between anemia and liver-associated disease severity, hepatic complications, and the progression of WD. RESULTS: A total of 288 WD patients (48 with and 240 without anemia) were enrolled in the study. Multivariate linear regression revealed that WD patients with anemia had significantly higher levels of bilirubin, alanine transaminase, prothrombin time, international normalized ratio, type Ⅳ collagen, and hyaluronic acid and significantly lower levels of albumin, total cholesterol, and high-density lipoprotein-cholesterol (all p < 0.05). Multivariate logistic regression showed that anemia was a risk factor for gastric varices and ascites (all p < 0.05). Fully adjusted Cox regression revealed that anemia was an independent risk factor for advanced Child-Pugh classification (p = 0.034). CONCLUSIONS: Anemia was common in WD patients and was associated with greater disease severity, a higher risk of hepatic complications, and a faster progression.

The efficacy of modified binding technique for renorrhaphy during robotic partial nephrectomy: surgical and functional outcomes from single-center experience.

BACKGROUND: To compare the traditional single-layer and double-layer suture renorrhaphy with modified "Binding" suture renorrhaphy (whole rim of the wound was closed by the all-layer flow suture starting from the parenchyma cut edges to hilum, followed by the final defect closure) in robotic partial nephrectomy (RPN) for treating localized renal cell carcinoma in our large institutional experience. METHODS: We retrospectively reviewed clinical data of 406 consecutive patients who underwent RPN from May 2018 and December 2020 in our center. The demographic and oncologic outcome variables were compared between different renal reconstruction groups and the effect of these suture techniques on renal function outcomes was also evaluated. RESULTS: For the single-layer group, median operative time and warm ischemic time were significantly less than that of the double-layer and "Binding" groups (p < 0.001), while the significantly lower eGFR drop (p = 0.014) was also detected within postoperative 3 months from baseline, but this difference lost its statistical significance from 3th month to the last follow-up. The changes in postoperative creatinine values were clinically insignificant among the three groups. In a sub-analysis over 258 patients with moderate/high nephrometry score, those patients who underwent "Binding" suture had an undifferentiated warm ischemic time, estimated blood loss, and length of hospitalization stay with a decreased risk of Grade III complications (postoperative hemorrhage requiring intervention) and improved renal function recovery during the whole follow-up. CONCLUSION: Single-layer suture renorrhaphy may be associated with better renal functional preservation and could prove to be reliable in patients with low-complexity tumor (RENAL score ≤ 6). Patients with moderate/high-complexity tumor (RENAL score ≥ 7) might represent a subgroup of patients having a functional benefit after "Binding" suture renorrhaphy even in the long-term period.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in Central Inner Mongolia, Northern China.

BACKGROUND: Cryptosporidium is a gastrointestinal protozoan that widely exists in nature, it is an established zoonotic pathogen. Infected cattle are considered to be associated with cryptosporidiosis outbreaks in humans. In the present study, we aimed to assess the prevalence and species distribution of Cryptosporidium in dairy cattle in Central Inner Mongolia. METHODS: We focused on the small subunit ribosomal RNA gene (SSU rRNA) of Cryptosporidium and 60-kDa glycoprotein gene (gp60) of Cryptosporidium parvum. We collected 505 dairy cattle manure samples from 6 sampling sites in Inner Mongolia in 2021; the samples were divided into 4 groups based on age. DNA extraction, polymerase chain reaction (PCR), sequence analysis, and restriction fragment length polymorphism (RFLP) using SspI and MboII restriction endonucleases were performed. RFLP analysis was performed to determine the prevalence and species distribution of Cryptosporidium. RESULTS: SSU rRNA PCR revealed that the overall prevalence of Cryptosporidium infection was 29.90% (151/505), with a prevalence of 37.67% (55/146) and 26.74% (96/359) in diarrheal and nondiarrheal samples, respectively; these differences were significant. The overall prevalence of Cryptosporidium infection at the 6 sampling sites ranged from 0 to 47.06% and that among the 4 age groups ranged from 18.50 to 43.81%. SSU rRNA sequence analysis and RFLP analysis revealed the presence of 4 Cryptosporidium species, namely, C. bovis (44.37%), C. andersoni (35.10%), C. ryanae (21.85%), and C. parvum (11.92%), along with a mixed infection involving two or three Cryptosporidium species. Cryptosporidium bovis or C. andersoni was the most common cause of infection in the four age groups. The subtype of C. parvum was successfully identified as IIdA via gp60 analysis; all isolates were identified as the subtype IIdA19G1. CONCLUSIONS: To the best of our knowledge, this is the first report of dairy cattle infected with four Cryptosporidium species in Inner Mongolia, China, along with a mixed infection involving two or three Cryptosporidium species, with C. bovis and C. andersoni as the dominant species. Moreover, this is the first study to identify C. parvum subtype IIdA19G1 in cattle in Inner Mongolia. Our study findings provide detailed information on molecular epidemiological investigation of bovine cryptosporidiosis in Inner Mongolia, suggesting that dairy cattle in this region are at risk of transmitting cryptosporidiosis to humans.

[Expression and significance analysis of GDF3 in testicular cancer based on TCGA and GTEx databases].

OBJECTIVE: To investigate the expression and significance of GDF3 in testicular cancer through bioinformatics analysis. METHODS: Using the TCGA and GTEx databases, differential expression analysis and pan-cancer analysis were performed to identify the target gene GDF3, and the clinical relevance of GDF3 in testicular cancer was analyzed using the UALCAN database. Based on the R packages "org.Hs.eg.db" and "clusterProfiler," gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to explore the potential functions of GDF3 in testicular cancer. The correlation of GDF3 with immune chemokines and immune inhibitors in testicular cancer was investigated using the TISIDB database. RESULTS: The GDF3 was significantly upregulated in testicular cancer (P<0.001) and closely associated with clinical staging (P<0.05) and tumor subtypes (P<0.001). The immune-related analysis revealed that GDF3 was strongly correlated with immune chemokines CCL26 (rho=0.599, P<0.001), CCL7 (rho=0.525, P<0.001), immune inhibitor ADORA2A (rho=0.723, P<0.001), and PVRL2 (rho=0.585, P<0.001). CONCLUSION: The GDF3 is closely related to the occurrence, development, and immune microenvironment of testicular cancer.

Photopolymerization-Driven Macroscopic Mechanical Motions of a Composite Film Containing a Vinyl Coordination Polymer.

We report a unique vinyl coordination polymer (CP), [Zn(4-Fb)2 (tkpvb)]n (1, 4-HFb=4-fluorobenzoic acid, tkpvb=1,2,4,5-tetrakis(4-pyridylvinyl)benzene) that undergoes a rare photopolymerization reaction to form a two-dimensional CP integrated with a one-dimensional linear organic polymer. Upon light irradiation at different wavelengths, 1 exhibits an unprecedented phenomenon of photoinduced nonlinear lattice expansion. 1 can be uniformly dispersed in polyvinyl alcohol (PVA) to form the composite film of 1-PVA. When this film is exposed to UV light, internal minute stresses within crystallites are released by lattice expansion, resulting in a variety of photopolymerization-driven macroscopic mechanical motions. The findings provide new insights into the conversion of small lattice expansions of CPs into macroscopic mechanical motions based on photopolymerization reactions, which can promote the development of CPs-based smart photoactuators in the burgeoning field of microrobotics.

Effect of ropivacaine combined with sufentanil epidural anesthesia in abdominal surgery.

To explore and analyze the effect of ropivacaine plus sufentanil for epidural anesthesia during abdominal surgery, a total of 120 patients who underwent abdominal surgery at our institution between May 2019 and November 2020 were recruited and randomly assigned at a 1:1 ratio to receive either ropivacaine alone for epidural anesthesia (control group) or ropivacaine plus sufentanil (observation group). The total anesthesia effect in the observation group was significantly higher than that in the control group (96.66% vs 78.33%) (P<0.05). The combined anesthesia resulted in significantly lower visual analogue scale (VAS) scores (1.51±0.84, 1.63±0.56, 1.69±0.63, 1.54±0.42) in patients at 4h, 8h, 16h and 24h postoperatively versus ropivacaine alone (2.35±0.88, 2.49±0. 69, 2.47±0.78, 2.39±0.58) (P<0.05). The Ramsay sedation score (RSS) scores (1.98±0.81, 2.44±0.62, 2.18±0.62, 2.51±0.37) of the observation group at 4h, 8h, 16h and 24h after operation were significantly lower than those of the control group (1.42±0.52, 1.73±0.71, 1.47±0.66, 1.68±0.62) (P<0.05). Patients receiving ropivacaine plus sufentanil were associated with a lower incidence of adverse reactions than those given ropivacaine only (5.00% vs 30.00%) (P<0.05). In abdominal surgery, ropivacaine plus sufentanil epidural anesthesia resulted in reduced postoperative pain, enhanced sedative effects and a lower risk of adverse reactions versus ropivacaine alone.

Molecular mechanism of CD44 homodimerization modulated by palmitoylation and membrane environments.

The homodimerization of CD44 plays a key role in an intercellular-to-extracellular signal transduction and tumor progression. Acylated modification and specific membrane environments have been reported to mediate translocation and oligomerization of CD44; however, the underlying molecular mechanism remains elusive. In this study, extensive molecular dynamics simulations are performed to characterize the dimerization of palmitoylated CD44 variants in different bilayer environments. CD44 forms homodimer depending on the cysteines on the juxta-membrane domains, and the dimerization efficiency and packing configurations are defected by their palmitoylated modifications. In the phase-segregated (raft included) membrane, homodimerization of the palmitoylated CD44 is hardly observed, whereas PIP2 addition compensates to realize dimerization. However, the structure of CD44 homodimer formed in the phase-segregated bilayer turns susceptive and PIP2 addition allows for an extensive conformation of the cytoplasmic domain, a proposal prerequisite to access the cytoskeleton linker proteins. The results unravel a delicate competitive relationship between PIP2, lipid raft, and palmitoylation in mediating protein homodimerization, which helps to clarify the dynamic dimer conformations and involved cellular signaling of the CD44 likewise proteins.

Substrate-Controlled Regioselectivity Switchable [3 + 2] Annulations To Access Spirooxindole Skeletons.

The additive-free [3 + 2] annulation from isatins, amino acids with 2-styrylbenzoxazoles, was described, providing a series of functional and structurally complex 3,3'-pyrrolidinyl-spirooxindole derivatives containing four contiguous and two quaternary stereogenic centers in high yields (up to 95%) and excellent diastereoselectivities (up to >25:1 dr). Interestingly, the reaction exhibits switchable regioselectivity depending on the substrate of amino acids. With proline or thioproline as the substrate, the reaction afforded α-regioselective spirooxindole skeletons. In contrast, when piperidine acid is the substrate, the reaction provided γ-regioselective spirooxindole skeletons.

Analgesic effects of different concentrations of ropivacaine in transversalis fascia plane block during laparotomy.

OBJECTIVE: To explore the analgesic effects of different concentrations of ropivacaine in transversalis fascia plane (TFP) block during laparotomy. METHODS: Ninety patients who underwent laparotomy admitted to our hospital from March 2019 to March 2020 were selected as the study subjects and were divided equally into a low concentration group, a medium concentration group, and a high concentration group according to the randomized grouping method. The low concentration group adopted 0.4% ropivacaine 40 ml, the medium concentration group was given 0.5% ropivacaine 40 ml, and the high concentration group was given 0.6% ropivacaine 40 ml. The hemodynamic indexes and the incidence of adverse reactions in the two groups were compared. The Numerical Rating Scale (NRS) was used to assess the postoperative pain in the three groups, the Bruggrmann comfort scale (BCS) was used to assess the comfort level in the three groups, and the Mini-mental State Examination (MMSE) was used to evaluate the postoperative cognitive function of the three groups of patients. RESULTS: The mean artery pressure (MAP) and heart rate (HR) levels at T1 and T2 were significantly lower in the medium concentration group than in the other two groups (P < 0.05). The low concentration group had a significantly higher NRS score at T2 than the medium concentration group and the high concentration group (P < 0.05). A significantly higher BCS score was observed in the high concentration group than the other two groups (P < 0.05). There were significantly higher Ramsay scores and MMSE scores in the medium concentration group than in the low concentration and high concentration groups (P < 0.05). The overall incidence of adverse reactions of the high concentration group was significantly higher than that of the low concentration group (P < 0.05), but showed similar results with the medium concentration group. CONCLUSION: The medium concentration group exhibits a better analgesic effect than the low concentration group and higher safety than the high concentration group. Therefore, the use of medium concentration ropivacaine in TFP block may provide a referential basis for clinical treatment.

Transcarotid transcatheter aortic valve replacement with cerebral embolic protection.

There remains concern about the risk of stroke with transcarotid transcatheter aortic valve replacement (TAVR). We describe the use of the Sentinel cerebral protection device with transcarotid TAVR in a patient at high risk for embolic complications. Deployment of the Sentinel filter in the innominate artery accommodated the insertion of the TAVR device through the bovine aortic arch via the contralateral carotid artery. By adapting the "flip-n-flex" technique from our previous transaxillary TAVR experience, we performed left transcarotid TAVR with the Sentinel device in an ergonomic manner and achieved coaxial placement of the transcatheter valve. Inspection of the Sentinel filter after the case revealed captured embolic debris.

Using aortic arch short axis views during transesophageal echocardiographic examination facilitates right ventricular assist device imaging.

Given the increased need for mechanical circulatory support and subsequent development of right ventricular assist devices (RVAD), appropriate imaging needs to be described to facilitate care in patients with cardiogenic shock and heart failure. We present three cases in which the upper esophageal aortic arch short axis (UE AA SAX) view on transesophageal echocardiography (TEE) was utilized to effectively image RVADs: to confirm normal positioning, to detect and guide repositioning, and to visualize malfunction. These cases support the importance of the UE AA SAX TEE view in RVAD outflow imaging and, when obtainable, should be included in routine RVAD assessment.

Identification of Quantitative Trait Locus and Candidate Genes for Drought Tolerance in a Soybean Recombinant Inbred Line Population.

With global warming and regional decreases in precipitation, drought has become a problem worldwide. As the number of arid regions in the world is increasing, drought has become a major factor leading to significant crop yield reductions and food crises. Soybean is a crop that is relatively sensitive to drought. It is also a crop that requires more water during growth and development. The aim of this study was to identify the quantitative trait locus (QTL) that affects drought tolerance in soybean by using a recombinant inbred line (RIL) population from a cross between the drought-tolerant cultivar 'Jindou21' and the drought-sensitive cultivar 'Zhongdou33'. Nine agronomic and physiological traits were identified under drought and well-watered conditions. Genetic maps were constructed with 923,420 polymorphic single nucleotide polymorphism (SNP) markers distributed on 20 chromosomes at an average genetic distance of 0.57 centimorgan (cM) between markers. A total of five QTLs with a logarithm of odds (LOD) value of 4.035-8.681 were identified on five chromosomes. Under well-watered conditions and drought-stress conditions, one QTL related to the main stem node number was located on chromosome 16, accounting for 17.177% of the phenotypic variation. Nine candidate genes for drought resistance were screened from this QTL, namely Glyma.16G036700, Glyma.16G036400, Glyma.16G036600, Glyma.16G036800, Glyma.13G312700, Glyma.13G312800, Glyma.16G042900, Glyma.16G043200, and Glyma.15G100700. These genes were annotated as NAC transport factor, GATA transport factor, and BTB/POZ-MATH proteins. This result can be used for molecular marker-assisted selection and provide a reference for breeding for drought tolerance in soybean.

Emerging Modulators of TMEM16A and Their Therapeutic Potential.

Calcium-activated chloride channels (CaCCs) are widespread chloride channels which rely on calcium activation to perform their functions. In 2008, TMEM16A (also known as anoctamin1, ANO1) was identified as the molecular basis of the CaCCs, which provided the possibility to study the physiological function of CaCCs. TMEM16A is widely expressed in various cells and controls basic physiological functions, including neuronal and cardiac excitability, nerve transduction, smooth muscle contraction, epithelial Cl- secretion and fertilization. However, the abnormal function of TMEM16A may cause a variety of diseases, including asthma, gastrointestinal motility disorder and various cancers. Therefore, TMEM16A is a putative drug target for many diseases, and it is important to determine specific and efficient modulators of TMEM16A channel. In recent years, we and others have screened several natural modulators of TMEM16A against cancers and gastrointestinal motility dysfunction. This article reviews the screening methods, efficacy of TMEM16A modulators and pharmacological effects of TMEM16A modulators on different diseases. GRAPHIC ABSTACT.

Activation of TMEM16A by natural product canthaxanthin promotes gastrointestinal contraction.

Transmembrane 16A (TMEM16A), also known as anoctamin 1, is the molecular basis of the calcium-activated chloride channels. TMEM16A is present in interstitial cells of Cajal, which are the pacemaker cells that control smooth muscle contraction. TMEM16A is implicated in gastrointestinal disorders. Activation of TMEM16A is believed to promote the gastrointestinal muscle contraction. Here, we report a highly efficient, nontoxic, and selective activator of TMEM16A, canthaxanthin (CX). The study using molecular docking and site-directed mutation revealed that CX-specific binging site in TMEM16A is K769. CX was also found to promote the contraction of smooth muscle cells in gastrointestinal tract through activation of TMEM16A channels, which provides an excellent basis for development of CX as a chemical tool and potential therapeutic for gastrointestinal dysfunction.

A meta-analysis of transcarotid versus transfemoral transcatheter aortic valve replacement.

BACKGROUND: Carotid access has shown promise as an excellent delivery route for transcatheter aortic valve replacement (TAVR). We aimed to compare outcomes of transcarotid (TC) and transfemoral (TF) TAVR by conducting a search and analysis of the best evidence in the literature to shed light on its safety and effectiveness. METHODS: The PubMed/MEDLINE, Embase, and Cochrane library from inception to July 2020 were searched to identify articles reporting comparative data on TC versus TF approaches for TAVR. Patients' baseline characteristics and clinical outcomes were extracted from the articles and pooled for analysis. RESULTS: Five studies, including a total of 2470 patients, were included in the study with 1859 patients in the TF group and 611 patients in the TC group. The TC group had higher prevalence of peripheral vascular disease, while the patients in the TF group was older. Meta-analysis revealed that there was no significant differences between the two groups with regard to 30-day mortality (p = 0.09), stroke (p = 0.28), new dialysis (p = 0.58), major bleeding (p = 0.69), or pacemaker implantation (p = 0.44). The TF group had a higher incidence of vascular complications (3.9% vs. 2.3%; OR 2.22; 95% CI [1.13, 4.38]; p = 0.02). CONCLUSIONS: Compared with the TF approach, TC-TAVR is associated with comparable procedural and clinical outcomes. Our analysis found a lower rate of vascular complication in TC access compared with TF access. This supports consideration of such an alternative access when there are concerns over the feasibility of TF access.

A copper iodide-catalyzed coupling reaction of benzofuran-3(2H)-ones with amines: an approach to α-ketoamides.

A CuI-catalyzed coupling reaction of benzofuran-3(2H)-ones with amines has been well established for the direct synthesis of α-ketoamides. This process involves C-O bond cleavage and C[double bond, length as m-dash]O/C-N bond formation. Mechanism studies indicated that this α-ketoamide formation reaction may involve a free radical process.

Left thoracotomy vs full sternotomy for centrifugal durable LVAD implantation: 1-year outcome comparison post-LVAD and post-heart transplantation.

Left ventricular assist device (LVAD) implantations have traditionally been approached through a full median sternotomy (FS). Recently, a minimally invasive left thoracotomy (LT) approach has been popularized. This study sought to compare the outcomes of FS and LT patients post-primary LVAD implantation and post-subsequent heart transplant (HT). This was a single-center retrospective study. 83 patients who underwent primary centrifugal durable LVAD implantation from January 2014 to June 2018 were included (FS, n = 41; LT, n = 42). 41 patients had a subsequent HT (FS, n = 19; LT, n = 22). Pre-operative patient demographics, intraoperative variables, post-operative 1-year survival, length of hospital stay, complications, and outcomes for LVAD implantation and following HT were analyzed. Intraoperative data showed that the LT group had a 23.4% longer mean LVAD implant surgical time (p < 0.01). One-year post-LVAD survival was similar between the two groups (p = 0.05). Complication rates, with the exception of the rate of hemorrhagic stroke (p = 0.04) post-LVAD implant were similar. One-year survival post-HT was similar between groups (p = 0.35). Complication rates and mean length of hospital stay were also similar (p = 1.0) post-HT. Our study demonstrated that LT approach does not negatively affect post-LVAD implantation or post-HT outcomes. Further, larger studies may determine more detailed effects of LT approach.