Variations in water use efficiency and carbon and nitrogen concentrations in red heart Chinese fir.

Temperature can significantly (P < 0.05) affect plant growth by modifying water use strategies, which are determined by intrinsic water use efficiency (WUEi). Red Heart Chinese Fir (Cunninghamia lanceolata) is one of the most important ecological and economic plantation species in China. However, the C. lanceolata water use strategy in response to increased temperatures and uneven temporal distribution of precipitation during the growing season is rarely reported. In a 7-year-old C. lanceolata plantation, differences in WUEi and C and N concentrations in different organs were analysed by anova, and the δ13C stable isotope, C, and N concentrations in stems determined at different tree heights. Stepwise regression and variance inflation factor were used to remove autocorrelated factors, and structural equation modelling was then used to explore relationships between WUEi and climate and biological factors. WUEi differed significantly between leaf and branch at different standardized precipitation evapotranspiration indices (SPEI). WUEi and N concentration decreased with age. The highest WUEi in branches and leaves were 92.7 and 88.4 µmol·mol-1 in 2020 (SPEI = 0.00), respectively. δ13C increased with relative tree height but N concentration and C/N ratio were not affected. Air temperatures has increased in between 2014 and 2020. WUEi and N concentration decreased with increasing branch and leaf age, but C concentration increased. SPEI significantly positively affected WUEi (P < 0.05), and WUEi was significantly negatively related to C concentration, which is consistent with the trade-off between C and water.

[Application of autogenous block bone graft in the reconstruction of severe alveolar crest defect].

Patients with severe alveolar ridge defects cannot be directly implanted and repaired, which seriously affects their quality of life. Onlay bone grafting is the main solution for severe alveolar ridge bone defect reconstruction, among which autogenous block bone grafting is the most widely used and is also the focus of clinical research on bone tissue reconstruction. This article expounds the characteristics and basic principles of autogenous bone block grafting, and comprehensively analyzes the selection of autogenous bone donor site, the principles of surgical operation, and the progress of bone graft techniques. In order to help surgeons make correct clinical decisions, increase the predictability of surgical effects, and improve the level of clinical diagnosis and treatment.

[A close contact of coronavirus disease 2019 with severe imported malaria: a case report].

This article presents a severe cerebral malaria patient in shock with a close contact of COVID-19 that was successfully cured in a negative pressure ward during the global pandemic of COVID-19. The patient experienced a sudden onset of high fever and coma in a designated isolation hotel after returning from Africa, and was transferred to a designated hospital. Following antimalarial therapy, blood pressure elevation, increase of blood volume, bedside hemodialysis, mechanical ventilation, plasma and platelet transfusions, the case gradual recovered.

[Study on related factors and characteristics of multimorbidity of overweight and obesity-related diseases in children in Hunan Province].

From January 2019 to December 2021, overweight and obese children who visited in health outpatient Center of Hunan Children's Hospital were studied to explore and analyze the rate, related factors and patterns of multimorbidity of overweight and obesity-related diseases in children in Hunan Province. Univariate and multivariate logistic regression models were used to analyze the multimorbidity-related factors of overweight and obesity-related diseases in children. Association rules (apriori algorithm) were used to explore the multimorbidity patterns of overweight and obesity-related diseases in children. A total of 725 overweight and obese children were included in this study. The multimorbidity rate of overweight and obesity-related diseases in children was 46.07% (334/725). Age, waist circumference, the frequency of food consumption such as hamburgers and fries and adding meals before bedtime were multimorbidity-related factors of overweight and obesity-related diseases in children. The multimorbidity associated with nonalcoholic fatty liver disease (NAFLD) was relatively common. The patterns with the top three support degrees were "NAFLD+dyslipidemia","NAFLD+hypertension" and "NAFLD+hyperuricemia". The patterns with the top three confidence and elevation degrees were "Hypertension+dyslipidemia => NAFLD","Hyperuricemia => NAFLD" and "NAFLD+hypertension => dyslipidemia".

Critical Non-Covalent Binding Intermediate for an Allosteric Covalent Inhibitor of SUMO E1.

Post-translational modifications by small ubiquitin-like modifiers (SUMOs) are dysregulated in many types of cancers. The SUMO E1 enzyme has recently been suggested as a new immuno-oncology target. COH000 was recently identified as a highly specific allosteric covalent inhibitor of SUMO E1. However, a marked discrepancy was found between the X-ray structure of the covalent COH000-bound SUMO E1 complex and the available structure-activity relationship (SAR) data of inhibitor analogues due to unresolved noncovalent protein-ligand interactions. Here, we have investigated noncovalent interactions between COH000 and SUMO E1 during inhibitor dissociation through novel Ligand Gaussian accelerated molecular dynamics (LiGaMD) simulations. Our simulations have identified a critical low-energy non-covalent binding intermediate conformation of COH000 that agreed excellently with published and new SAR data of the COH000 analogues, which were otherwise inconsistent with the X-ray structure. Altogether, our biochemical experiments and LiGaMD simulations have uncovered a critical non-covalent binding intermediate during allosteric inhibition of the SUMO E1 complex.

Flight hormones as therapeutic target for novel Coronavirus infectious disease.

Coronavirus Disease 2019 (COVID-19) pandemic has made more awful effect on wellbeing and economy worldwide on an extraordinary scale. Angiotensin I Converting Enzyme 2 (ACE2), the principal receptor of SARS-CoV2, has been found to be communicated with Dopa decarboxylase in unwinding the connection of catecholamines with COVID-19 infection. Cardiovascular (CV) sickness, diabetes, hypertension, and related conditions cause significant risks during the current situation and the affected people are under basic observation around the world. The hypertension and diabetes are related with alterations in the degrees of catecholamines associated with renal gland. The naive form of renal dopaminergic framework is related with the expanded reabsorption of sodium resulting in downregulation of the ACE2 expression. Catecholamine biosynthesis is managed by counter-controlling angiotensin type 1R (AT1R) and angiotensin type 2R (AT2R), incitement of AT2 lessens catecholamine biosynthesis by means of a diminishing in cGMP levels likewise incitement of AT1 initiate catecholamine biosynthesis. This audit sums up the conceivable contribution of catecholamines in intense COVID-19 contamination and furthermore featured possible restorative adequacy of catecholamine flagging pathways against the incessant SARS-CoV-2.

Higher frequency electrical stimulation enhanced myloglossus satellite cell differentiation by upregulating expression of Pax7 mRNA, MyoD, myogenin and MyHC protein.

OBJECTIVE: We investigated the effect of electrical stimulation (ES) of varying pulse frequency on differentiation and proliferation of canine myloglossus satellite cells in vitro. MATERIALS AND METHODS: Cellular viability and proliferation were assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometry fluorescence-activated cell sorting analysis. Cellular differentiation and expression of mark molecule were assayed by Real Time-PCR and Western blot. RESULTS: With increasing frequency ES, we found a significant increase in Myod (r=0.988, p<0.0001), myogenin (r=0.988, p<0.0001), MyHC-slow (r=0.988, p<0.0001), MyHC-fast (r=0.875, p<0.0001) protein expression, and Pax7 mRNA expression (r=0.712, p=0.001). CONCLUSIONS: Pax7 mRNA expression and MyoD, myogenin, and MyHC protein expression were increased with increment of electrical stimulation frequency in myloglossus muscle satellite. Higher frequency ES enhanced myloglossus satellite cell differentiation, not proliferation and viability.

LncRNA H19 promotes glioma angiogenesis through miR-138/HIF-1α/VEGF axis.

Glioma is one of the most common and aggressive malignant primary brain tumors with high recurrence rate and mortality rate and heavily depends on the angiogenesis. LncRNA H19 has many diverse biological functions, including the regulation of cell proliferation, differentiation and metabolism. Here, we aimed to investigate the molecular mechanism of lncRNA H19 affecting angiogenesis in glioma, which could help to uncover potential target for glioma therapy. RT-qPCR analysis was performed to detect the expression of lncRNA H19 and miR-138 in HEB, U87, A172 and U373 cell lines. MTT assay was used to evaluate the cell viability. To evaluate the migration and invasion after lncRNA H19 knockdown, Transwell and wound healing assay were employed. After lncRNA H19 knockdown, protein expression of HIF 1α and VEGF was detected by western blot and tube formation was assessed. For the prediction and validation of the interaction between lncRNA H19 and miR-138, bioinformatics and luciferase assay were performed. We investigated the regulatory roles and downstream molecular mechanisms of lncRNA H19 in glioma by knockdown H19, which inhibited the proliferation, migration and angiogenesis of glioma cells. Moreover, miR-138 acted as a target of H19 as detected by luciferase reporter assays. Meanwhile, HIF-1α was also a target of miR-138 and miR-138 could also regulate the proliferation, migration and angiogenesis of glioma cells by targeting HIF-1α and affecting the expression of VEGF in turn. Our findings identified an upregulated lncRNA H19 in glioma cells, which could promote proliferation, migration, invasion and angiogenesis via miR-138/HIF-1α axis as a ceRNA. This study provided a new opportunity to advance our understanding in the potential mechanism of lncRNA in glioma angiogenesis.

Curcumin inhibits hypoxia inducible factor-1α-induced inflammation and apoptosis in macrophages through an ERK dependent pathway.

OBJECTIVE: Atherosclerosis, a kind of peripheral arterial disease with chronic inflammation, leads to the dysfunction of the vascular system and many other diseases. Hypoxia has been proven to participate in the progression of atherosclerosis, while curcumin can inhibit hypoxia-inducible factor 1α (HIF-1α). However, the underlying mechanisms are still elusive. PATIENTS AND METHODS: qRT-PCR was used to examine the expression of HIF-1α, IL-6 and TNFα of macrophages under hypoxic condition. Western blot was applied to examine the changes of HIF-1α, ERK and p-ERK after treatment with curcumin. Oli Red O staining and enzymatic assay were used to examine the lipid and total cholesterol in macrophages, respectively. ELISA was used to examine the release of IL-6 and TNFα by macrophages. FACS and MTT assays were applied to examine the apoptosis and proliferation of macrophages. RESULTS: Here, we found curcumin inhibited the expression of HIF-1α at the protein level in macrophages under hypoxic condition and curcumin and HIF-1α inhibitors repressed the total cholesterol and lipid level in macrophage under hypoxic condition. Moreover, curcumin also decreased the expression of HIF-1α downstream genes, VEGF, HMOX1, ROS and PDGF. Then, the data show the HIF-1α-induced apoptosis and inflammation of macrophages were inhibited by curcumin. Curcumin also rescued the proliferation defect of macrophages caused by hypoxia. Furthermore, we found it inhibited the expression of HIF-1α via ERK signaling pathway. CONCLUSIONS: We describe that curcumin inhibited the HIF-1α-induced apoptosis and inflammation of macrophages via ERK signaling pathways. These results suggest curcumin can be used for the treatment of atherosclerosis.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme.

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

[Circular RNA circHIPK3 acts as the sponge of microRNA-124 to promote human oral squamous cell carcinoma cells proliferation].

Objective: To explore the expression and clinical significance of circular RNA circHIPK3 in oral squamous cell carcinoma (OSCC), analyze the effect of circHIPK3 on the proliferation of OSCC cells. Methods: The expression of circHIPK3 in OSCC tissues, adjacent non-cancerous tissues and OSCC cell lines were detected by quantitative real-time polymerase chain reaction (qPCR). The correlations between the expression of circHIPK3 in OSCC tissues and the clinicopathological features were analyzed as well. circHIPK3-specific siRNA si-circHIPK3 and negative control siRNA si-NC were designed and synthesised and used to transfect CAL27 and SCC15 cells respectively. The proliferation capacity of CAL27 and SCC15 cells after transfection with si-circHIPK3 was detected by CCK-8 assay. The expression of miR-124 in OSCC was detected by qPCR, and the correlation between expression of circHIPK3 and the expression of miR-124 was analyzed. Using qPCR to detect the expression of miR-124 in CAL27 and SCC15 cells after transfection with si-circHIPK3 and si-NC respectively. Furthermore, using CCK-8 assay to detect the proliferation capacity of CAL27 and SCC15 cells after transfection with si-NC, si-circHIPK3, miR-124 mimic, si-circHIPK3+miR-124 inhibitor. Results: The expression of circHIPK3 in OSCC tissues [2.23 (1.86, 3.00)] was significantly higher than that of the adjacent non-cancerous tissues [1.05 (0.85, 1.26)] (U=1 094, P=0.000). The expression of circHIPK3 in CAL27 (3.02±0.51) and SCC15 cells (3.16±0.75) was higher than those of human normal oral keratinocytes (hNOK) (1.26±0.30) (P=0.000). The expression of circHIPK3 was found to be closely associated with TNM stage (P<0.05) and tumor grades (P<0.05). Knockdown of circHIPK3 can inhibit proliferation of CAL27 and SCC15 cells (P<0.05). The expression of miR-124 in OSCC tissues (0.61±0.35) was significantly lower than that in adjacent non-cancerous tissues (1.13+0.39) (t=-5.36, P<0.05). Correlation analysis showed that the expression of circHIPK3 in OSCC was negatively correlated with the expression of miR-124 (r=-0.767, P<0.001). Moreover, down-regulation of miR-124 rescued the phenotype induced by knockdown of circHIPK3. Conclusions: The expression of circHIPK3 in OSCC was increased, and silencing of circHIPK3 expression can inhibit the proliferation of OSCC cells. Our results suggest that circHIPK3 may play a key role in the occurrence and development process of OSCC through the regulation of miR-124 expression.

Endothelial progenitor cell miR-126 promotes homing of endothelial progenitor cells within arterial thrombus in patients with cerebral infarction and its molecular mechanism.

OBJECTIVE: The present study was designed to investigate the effect of microRNA-126 (miR-126) on the migration and homing of endothelial progenitor cells (EPCs) within arterial thrombus of cerebral infarction patients. MATERIALS AND METHODS: EPCs from rat bone marrow were isolated, and miR-126 overexpressed EPCs were constructed by lentiviral transfection. Then, the middle cerebral artery occlusion (MCAO) model was established by the method of thread ligation. Successfully established model rats were randomly divided into miR-126 overexpression EPC group, miR-126 wild type EPC group, and normal saline group. One day after the infarction, the miR-126 overexpression EPCs, miR-126 wild type EPCs, and normal saline, were injected into the lateral ventricle of the corresponding groups. Also, the transplanted cells were tracked by cell dye SPDiIC18. The expression of tight junction proteins ZO-1 and Claudin-5 in brain tissue was detected by Western blotting. RESULTS: Transplanted cells were detected in the cerebral infarction area 3 days after transplantation by cell dye SP-DiIC18. The number of homing EPCs in miR-126 overexpression group was significantly higher than that of miR-126 wild type EPC group (p < 0.05). Also, the protein expression of ZO-1 and Claudin-5 in the miR- 126 overexpression EPC group was significantly higher compared with that of the miR-126 wild type EPC group and the normal saline group. CONCLUSIONS: miR-126 overexpression EPCs, which were transplanted in the lateral ventricle, can home to the cerebral infarction areas via increasing increase.

Norepinephrine and acetylcholine changes during electrically-induced atrial fibrillation episodes in canine models.

Atrial fibrillation (AF) is the most prevalent heart rhythm disorder, and autonomic nervous system (ANS) is important to AF. This study aims to identify whether changes in transmitters released by ANS could reflect their activities. The right atrium (RA) groups (1-40V) included RA500 and RA1000. While ANS groups received high-frequency electrical stimulation (1-8V, 20 Hz, 2 ms), including left stellate ganglion stimulation (LSGS) andleft cervical vagus trunk stimulation (LVTS). The induced rate of AF, duration and atrial effective refractory period (AERP) were measured. The blood was drawn for evaluation of norepinephrine (NE) and acetylcholine (Ach) concentrations. At 12-hours, RA tissue was dissected and compared against un-stimulated controls. While AF was induced by all groups, duration and AERP were significantly different between RA pacing groups and ANS-stimulated groups, respectively (P<0.05). Specific changes in profile of NE and Ach were associated with modality of stimulation. RA1000 tended to display most significant changes (P<0.05) compared to other groups while variables concentration levels were observed in other groups. In conclusion, electrically-induced AF initiated by various modalities of stimulation showed different changes in serum and RA tissues. Fast frequency pacing caused significant atrial electrical remodeling, including ANS activity change.

Structure of A-C Type Intervariant Interface in Nonmodulated Martensite in a Ni-Mn-Ga Alloy.

The structure of A-C type intervariant interface in nonmodulated martensite in the Ni54Mn25Ga21 alloy was studied using high resolution transmission electron microscopy. The A-C interface is between the martensitic variants A and C, each of which has a nanoscale substructure of twin-related lamellae. According to their different thicknesses, the nanoscale lamellae in each variant can be classified into major and minor lamellae. It is the boundaries between these lamellae in different variants that constitute the A-C interface, which is thus composed of major-major, minor-minor, and major-minor lamellar boundaries. The volume fraction of the minor lamellae, λ, plays an important role in the structure of A-C interfaces. For major-major and minor-minor lamellar boundaries, they are symmetrical or asymmetrical tilt boundaries; for major-minor boundary, as λ increases, it changes from a symmetrical tilt boundary to two asymmetrical microfacets. Moreover, both lattice and misfit dislocations were observed in the A-C interfaces. On the basis of experimental observations and dislocation theory, we explain how different morphologies of the A-C interface are formed and describe the formation process of the A-C interfaces from λ ≈ 0 to λ ≈ 0.5 in terms of dislocation-boundary interaction, and we infer that low density of interfacial dislocations would lead to high mobility of the A-C interface.

[Clinical research of differences of volume of pharyngeal cavity between normal healthy subjects and patients with severe obstructive sleep apnea hypopnea syndrome].

Objective:To investigate the differences of volume of pharyngeal cavity between normal healthy subjects and patients with severe obstructive sleep apnea hypopnea syndrome(OSAHS)before and after surgery and its application value in assessment of surgical curative effect.Method:Forty-four healthy male adults were included in normal group.Thirty-eight patients with severe OSAHS diagnosed by polysomnography were included in experimental group(OSAHS group). Volume of pharyngeal cavity, oropharynx and laryngopharynx measured by acoustic pharyngealmetry and AHI, obstructive apnea frequency and the lowest oxygen saturation recorded by PSG monitor were all obtained in normal groups and experimental groups before and 3 months after the surgery. All the data are statistically analyzed. Result:Volume of pharyngeal cavity,AHI,obstructive apnea frequency and lowest oxygen saturation of patients in experimental group were all improved after surgery compared with pre-operation(P<0.01). Conclusion: The measurement of pharyngeal cavity volume is helpful to the quantitative analysis of upper airway structure and determine the stenosis location, it is also useful to guide surgery and evaluate the curative effect of the surgery.

Genetic affinities of central China populations.

Hunan locates in the south-central part of China, to the south of the middle reaches of the Yangtze River and south of Lake Dongting. According to the historical records, the peopling of Hunan by modern human ancestors can ascend to 40 thousand years ago. Thus, to trace the ancient maternal components can offer further insight into the origin of south-central China. In this study, we investigated the mitochondrial DNA of 114 individuals from Hunan Province (including 34 Han, 40 Tujia and 40 Miao). Hypervariable regions I and II of the mtDNA control region were sequenced, and the relative diagnostic variations in coding region according to the updated worldwide phylogeny tree were selected and typed by restriction fragment length polymorphism analysis or direct sequencing. All individuals were classified into specific (sub)haplogroups. By comparison with the surrounding populations, southern China-prevalent haplogroups were detected with relative higher frequency in the Tujia and Miao ethnic populations, such as haplogroup B, with more than 20%, lacking in the Han population, which illustrated its southern origin characters. In addition, we also detected northern of East Asia prevalent haplogroups with a relative higher frequency in Tujia populations than in the Miao and Yao ethnic groups, implying a gene flow from Han populations. However, the language-clustering tendency was supported by our principal component analysis and further genetic estimation results. Han and ethnic groups in central China exhibited specific ancestors related to their closer language affinity, although there was extensively genetic admixture between Han and ethnic groups.

Serum IL-21 levels decrease with glucocorticoid treatment in myasthenia gravis.

This study aimed to observe the changes of IL-21/21R in peripheral blood of myasthenia gravis (MG) patients with glucocorticoid treatment and further clarify its role in pathogenesis of MG. 20 MG patients and 15 healthy controls were enrolled in this prospective study. Measurement of serum IL-21 concentration in healthy controls and MG patients before and after glucocorticoid treatment was done using ELISA, whereas expression of IL-21R mRNA was determined by RT-PCR. In addition, serum levels of specific anti-AChR-IgG and its subclasses IgG1, IgG2, IgG3 were also determined by ELISA as follows: (1) serum IL-21 concentration in MG patients was higher before treatment 86.94 ± 14.47 (pg/ml) and decreased significantly after glucocorticoid treatment 35.84 ± 16.13 (pg/ml) (p < 0.05), (2) relative OD values of IL-21R mRNA expressed in PBMCs of MG patients was higher 0.137 ± 0.023 and significantly decreased after glucocorticoid treatment 0.114 ± 0.023 (p < 0.05), while it was 0.107 ± 0.025 in control group, (3) serum concentration of IL-21 showed positive correlation with specific serum anti-AchR-IgG1 levels. The results indicate that the serum IL-21 decreases with glucocorticoid treatment and might be crucial in pathogenesis of mechanism of glucocorticoid treatment on MG patients.

Cancer-initiating cells derived from human rectal adenocarcinoma tissues carry mesenchymal phenotypes and resist drug therapies.

Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial-mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

Prenatal diagnosis of criss-cross heart: sonographical and pathological features of five cases.

OBJECTIVE: To describe the sonographical and pathological features of fetal criss-cross heart (CCH). STUDY DESIGN: All cases of fetal CCH diagnosed by fetal echocardiogram from May 2003-May 2011 were identified at a single referral center using an established perinatal database. Demographic and genetic information, sonographical images and autopsy reports were reviewed. Sonographical and pathological features are described. RESULT: Five cases of fetal CCH were identified, all of which were confirmed by autopsy. Characteristic sonographical findings include: (1) the inability to obtain four-chamber view at standard transverse plane through the fetal chest; (2) appreciation of the misaligned spatial atrial-ventricle connection with the interventricular septum in a 'spiraling' orientation; (3) orientation of the two ventricular inlets in a superior-inferior and crossing position; and (4) a four-chamber-like view seen in the sagittal plane of the fetal chest. Doppler ultrasound demonstrates the 'criss-cross' arrangement of the inflow tracts into the two ventricles simultaneously in the transverse plane of the fetal chest. CONCLUSION: CCH is a rare developmental disorder that can be accurately diagnosed prenatally. Early diagnosis will allow for more targeted counseling and early intervention.