CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.

Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: • Rapid marker recycling based on the visualization of a green fluorescent protein • One-step multifragment integration and large fragment knockout in the genome • A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.

The Tumor-Immune Response Is Not Compromised by Mesenchymal Stromal Cells in Humanized Mice.

Therapeutic uses of mesenchymal stromal cells (MSCs) have emerged over the past decade. Yet, their effect on tumor growth remains highly debated, particularly in an immune competent environment. In this study, we wanted to investigate the impact of human umbilical cord-derived MSCs (hUC-MSCs) on tumor growth in humanized mice generated by the human adoptive transfer of PBMCs or the cotransplantation of hematopoietic stem cells and human thymic tissue (human BLT [Hu-BLT]). Our results showed that the growth and immune rejection of engineered human fibroblastic tumors was not altered by the injection of hUC-MSCs in immune-deficient or humanized mice, respectively. This was observed whether tumor cells were injected s.c. or i.v. and independently of the injection route of the hUC-MSCs. Moreover, only in Hu-BLT mice did hUC-MSCs have some effects on the tumor-immune infiltrate, yet without altering tumor growth. These results demonstrate that hUC-MSCs do not promote fibroblastic tumor growth and neither do they prevent tumor infiltration and rejection by immune cells in humanized mice.

Sedimentary organic carbon and nutrient distributions in an endorheic lake in semiarid area of the Mongolian Plateau.

Due to the lack of outlets, inflowing pollutants are often deposited in an endorheic lake, posing potential pressure on the environment. With climate change, extreme weather is expected to be more frequent and will contribute to the release of carbon and nutrients buried in the lakebeds. However, the distribution of sedimentary organic carbon and nutrients and the mechanisms that control the distribution are not fully understood, despite their significance to environmental development in endorheic lakes being widely recognized. In this study, the mechanisms controlling the sedimentary organic carbon and nutrient distributions in endorheic lakes were examined based on the analysis of an endorheic lake in the semiarid area of the Mongolian Plateau. The field survey results indicate that the concentrations of sedimentary organic carbon (TOC) and nutrients (NH3-N and TP) on the lakebed have significant correlations and present spatial heterogeneities. To further study the distribution mechanisms, numerical models were established to calculate the age of the water discharged from the rivers around the lake, and satellite remote sensing data were applied to examine the external source of organic carbon and nutrients and the factors influencing their movements to the lake. Based on the distribution of the water age, the water flow and mass transport trends in Lake Hulun were determined, and the time scales of the environmental processes were compared with those of water circulation. Further analysis indicates that the water circulation in the lake favors the accumulation of sedimentary organic carbon and nutrients in the northwestern part of the lake, and the organic carbon produced in the lake is transported to this region within an ice-free period. Satellite remote sensing data indicate that the region on the northwest bank of the lake experiences a larger terrestrial slope and better vegetation coverage than that on the southeast bank, which corresponds to a higher concentration of sedimentary organic carbon and nutrients in the northwest of the lake. This suggests that the sediment quality is closely related to the environment around the endorheic lake, and the larger slope and better vegetation coverage are significant factors for the high concentration of sedimentary organic carbon and nutrients on the lakebed under the conditions of scarce precipitation and low temperature. This study provides a theoretical basis and direction for further protection and management of the ecological environment of endorheic lakes.

Penisarins A and B, Sesquiterpene Coumarins Isolated from an Endophytic Penicillium sp.

Penisarins A (1) and B (2), sesquiterpene coumarins with an unusual tricyclic sesquiterpene system, were isolated from endophytic Penicillium sp. KMU18029. Their structures were elucidated on the basis of spectroscopic methods, single-crystal X-ray diffraction, and electronic circular dichroism calculations. Compound 2 showed significant cytotoxicities against two human cancer cell lines, HL-60 and SMMC-7721, with IC50 values of 3.6 ± 0.2 and 3.7 ± 0.2 µM, respectively.

Microfibril-Associated Protein 2 (MFAP2) Potentiates Invasion and Migration of Melanoma by EMT and Wnt/ß-Catenin Pathway.

BACKGROUND Growing evidence indicates an association between microfibril-associated protein 2 (MFAP2) and a number of physiological and pathological mechanisms. The potential role of MFAP2 in cancer requires further elucidation. The present study investigated the biological behavior of MFAP2 in melanoma patients. MATERIAL AND METHODS MFAP2 inhibition was established in the B16 melanoma cell line through the use of RNA interference and was assessed by quantitative real-time PCR (qRT-PCR) and Western blot analysis. Wound-healing analysis, transwell assay, and in vivo imaging were performed to investigate the roles of MFAP2 reducing cell mobility, migration, and invasion abilities in vitro and in vivo. RESULTS We found substantially higher MFAP2 expression in B16 melanoma cells. The knockdown of MFAP2 inhibited B16 melanoma cells migration and invasion. Western blot analysis was used to assess changes in biomarkers of EMT, indicating the function of MFAP2 in EMT. We found that downregulation of MFAP2 altered the expression of Wnt/ß-catenin-linked protein. CONCLUSIONS Our results suggest that MFAP2 has potential as a molecular target to treat melanoma and suppress metastasis of melanoma cells.

TD-DFT Study on Thiacalix[4]arene, the Receptor of a Fluorescent Chemosensor for Cu2.

The electronic and spectral properties of p-tert-butylthiacalix[4]arene (TCA) and its Cu2+ complexes (CuTCA and Cu2TCA) were characterized by time-dependent density functional theory (TD-DFT). Geometries of TCA, CuTCA, and Cu2TCA were optimized at the CAM-B3LYP/6-31+G(d,p) level of theory in water using the conductor-like polarizable continuum model. The absorption spectra of TCA, CuTCA, and Cu2TCA were demonstrated by TD-DFT method. The quenching mechanism of perylene as the fluorophore of a chemosensor based on thiacalix[4]arene was discussed. The addition of Cu2+ to TCA introduced a series of low-lying excited states involving copper d orbitals. The overlap between absorption of TCA complexes and emission of perylene indicated that the quenching of perylene fluorescence is due to fluorescence resonance energy transfer.

Detection of defects on the surface of a semiconductor by terahertz surface plasmon polaritons.

We propose a new method for detecting small defects on the surface of a semiconductor by analyzing the transmission spectrum of terahertz surface plasmon polaritons. The field distributions caused by the detection of defects of different sizes are simulated. Experimentally, using a terahertz time domain spectrometer, we measure the transmission spectrum of terahertz surface plasmon polaritons passing through particles on the surface of an intrinsic InSb wafer. Our results show that the measured temporal waveform and frequency spectra are distinctly changed due to the presence of the particles, thereby confirming the effectiveness of this method for detecting defects. For increased detection efficiency, the frequency of the surface plasmon polaritons has to be slightly lower than the plasma frequency of the semiconductor. In comparison with traditional methods, our approach offers the merits of detecting both on-surface and subsurface defects, which is critical in monitoring the quality of semiconductor wafers.

BCL2 promotor methylation and miR-15a/16-1 upregulation is associated with sanguinarine-induced apoptotic death in rat HSC-T6 cells.

Previous studies show that several pathways are involved in sanguinarine-induced apoptotic cell death, including AKT downregulation, inhibition of NF-kB activation, mediation of ROS production, downregulation of anti-apoptosis proteins XIAP and cIAP-1, upregulation of BAX, and downregulation of BCL2. In this study, we found out that the quenching of ROS generation by N-acetyl-l-cysteine (NAC), a scavenger of ROS, reversed sanguinarine-induced apoptosis effects, also we found out that sanguinarine-induced rat hepatic stellate T6 cells (HSC-T6 cells) apoptosis was correlated with the generation of increased ROS, which was followed by the activation of caspase-8 (-3, -6, and -9), and the decreasing in the miltochondrial membrane potential (MMP) and the down-regulation of anti-apoptotic protein Bcl-2. It is not clear whether BCL2's downregulation relates to its promoter methylation and miR-15a/16-1 expression which can bind to BCL2 3'-UTR (un-translation reagon). We showed that sanguinarine-induced down regulation of BCL2 was associated with the increased methylation rate of BCL2 promotor district and the increased expression of miR-15a/16-1. HSC-T6 cells treatment with 5-Aza-2'-deoxycytidine (5'-Aza-CdR) impeded sanguinarine-induced BCL2 promotor district methylation and recovered BCL2's expression. Over expression of BCL2 using pEGFP-N1 vector decreased sanguinarine-induced HSC-T6 cells apoptotic death significantly but not completely. These observations clearly showed that BCL2 down regulation was associated with its promoter methylation and miR-15a/16-1 upregulation in sanguinarine-induced Rat HSC-T6 cells.

Developmental role of macrophages modeled in human pluripotent stem cell-derived intestinal tissue.

Macrophages populate the embryo early in gestation, but their role in development is not well defined. In particular, specification and function of macrophages in intestinal development remain little explored. To study this event in the human developmental context, we derived and combined human intestinal organoid and macrophages from pluripotent stem cells. Macrophages migrate into the organoid, proliferate, and occupy the emerging microanatomical niches of epithelial crypts and ganglia. They also acquire a transcriptomic profile similar to that of fetal intestinal macrophages and display tissue macrophage behaviors, such as recruitment to tissue injury. Using this model, we show that macrophages reduce glycolysis in mesenchymal cells and limit tissue growth without affecting tissue architecture, in contrast to the pro-growth effect of enteric neurons. In short, we engineered an intestinal tissue model populated with macrophages, and we suggest that resident macrophages contribute to the regulation of metabolism and growth of the developing intestine.

Interaction of glycine with Li+ in the (H2O)n (n = 0-8) clusters.

CONTEXT: We investigated the interaction between glycine and Li+ in water environment based on the Gly·Li+(H2O)n (n = 0-8) cluster. Our study shows that for Gly·Li+, Li+ binds to both carbonyl oxygen and amino nitrogen to form a bidentate structure, and the first three water molecules preferentially interact with Li+. For n = 0-5, the complexes of Gly·Li+(H2O)n exist in neutral form, and when the water number reached 6, the complex can coexist in neutral and zwitterionic form, then zwitterionic structures are dominant for n = 7, 8. The analyses by RDG, AIM, and ESP in conjunction with the calculated interaction energies show that the interaction between Li+ and Gly decreases gradually with the water molecules involved successively from n = 1 to 6 and then increases for n = 7-8. Additionally, the infrared spectra of Gly·Li+(H2O)n (n = 0-8) are also calculated. METHODS: The initial structures were optimized using Gaussian 09 program package in B3LYP-D3 (BJ)/6-311G(d, p) method, and the frequency was calculated with 6-311 + G(2d, p) basis set. GaussView5.0.9 was used to view simulation infrared spectra. The noncovalent interaction method (NCl), energy decomposition (EDA), atoms in molecules (AIM) analysis, and electrostatic potential (ESP) analyses were conducted using Multiwfn software to gain a deeper understanding of the interaction properties of Gly, Li+, and water.

Preparation and characterization of glucoamylase microcapsules prepared by W/O/W type complex coacervation freeze drying.

Glucoamylase was often used in the brewing industry but was unstable to several environmental factors and reacted quickly to produce fermentable sugar, which limited its applications. Microencapsulation could effectively overcome the drawbacks. This study evaluated the feasibility of the preparation of glucoamylase microcapsules (GM) using W/O/W complex coacervation-freeze-drying method. The parameters of the microcapsules were optimized by the response surface optimization design: core-wall ratio at 1:1, wall-material concentration at 8%, and coagulation time for 20 min. Under current condition, the final microencapsulation efficiency reached 85.64 ± 1.60%. Glucoamylase could be slowly released for more than 96 h in the liquid state, and could react slowly for more than 336 h in the solid state. The optimized GM were incubated for 1 h, and the relative enzyme activity was better than that of free glucoamylase under high temperature conditions. The water capacity, solubility, morphology, differential scanning calorimetry, and Fourier transform infrared spectroscopy were conducted. Glucoamylase exhibited good sustained release characteristics. The microcapsules were more resistant to environmental stimuli and showed stronger robustness after optimization. PRACTICAL APPLICATION: Saccharification enzymes are often used in the winemaking industry, and direct addition will cause the fermentation process to heat up too quickly, resulting in the inactivation of microorganisms and saccharification enzymes, affecting the efficiency of saccharification enzymes. Therefore, microcapsules are used to encapsulate the saccharification enzyme, and its efficacy is slowly released for a long time during the fermentation process.

Immobilized glucoamylase based on ZIF-8: Preparation, response surface optimization, characterization.

The glucoamylase@ZIF-8 was prepared using ZIF-8 material as the carrier in this study. The preparation process was optimized by response surface methodology, and the stability of glucoamylase@ZIF-8 was determined. The material was characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The results showed that the optimum preparation process of glucoamylase@ZIF-8 was 1.65 mol 2-methylimidazole, 5.85 mL glucoamylase, 33°C stirring temperature, 90 min stirring time, and 84.0230% ± 0.6006% embedding rate. At 100°C, the free glucoamylase completely lost its activity, whereas the glucoamylase@ZIF-8 still had a retained enzyme activity of 12.0123% ± 0.86158%; at pH 3-6, the highest activity of glucoamylase@ZIF-8 was 95.9531% ± 0.96181%, and about 80% of glucoamylase activity could be retained under alkaline conditions. When the ethanol concentration was 13%, the retained enzyme activity was 7.9316% ± 0.19805%, significantly higher than free enzymes. The Km of glucoamylase@ZIF-8 and free enzyme were 1235.6825 and 80.317 mg/mL, respectively. Vmax was 0.2453 and 0.149 mg/(mL min), respectively. The appearance, crystal strength, and thermal stability of glucoamylase@ZIF-8 were improved after optimization, and they had high reusability.

Chemosensory Characteristics of Brandies from Chinese Core Production Area and First Insights into Their Differences from Cognac.

This work aimed to compare the aroma characteristics of representative brandies with different grades from Yantai (one of the Chinese core production areas) and Cognac and to establish relationships between sensory descriptors and chemical composition. Descriptive analysis was performed with a trained panel to obtain the sensory profiles. Forty-three aroma-active compounds were quantified by four different methodologies. A prediction model on the basis of partial least squares analysis was performed to identify candidate compounds that were unique to a certain group of brandies. The result showed that brandies from Yantai could be distinguished from Cognac brandies on the basis of spicy, dried fruit, floral, and fruity-like aromas, which were associated with an aromatic balance between concentrations of a set of compounds such as 5-methylfurfural, γ-nonalactone, and γ-dodecalactone. Meanwhile, brandy with different grades could be distinguished on the basis of compounds derived mostly during the aging process.

KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.

Introduction: Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function. Methods: To restore NK cell cytotoxicity against HLA-E+ tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated KLRC1 gene editing. Results: KLRC1 knockout resulted in a reduction of 81% of NKG2A+ cell frequency in ex vivo expanded human NK cells post-cell sorting. In vitro, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with p-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, KLRC1 KO NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E+ solid tumor cell lines, with p-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A- NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in KLRC1 KO NK cells and contributed to the improved NK cell cytotoxicity against HLA-E+ tumors. In vivo, the adoptive transfer of human KLRC1 KO NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E+ metastatic breast cancer, as compared to WT NK cells (p = 0.0015). Conclusions: Our results demonstrate that KLRC1 knockout is an effective strategy to improve NK cell antitumor activity against HLA-E+ tumors and could be applied in the development of NK cell therapy for solid tumors.

Estimation of nutrient (N and P) fluxes into Newark Bay, USA.

This study was conducted in northern New Jersey, USA, to estimate the nutrient fluxes from the Passaic River, the Hackensack River and other sources into Newark Bay and the nutrient residence time in Newark Bay. Bi-weekly total inorganic nitrogen (TIN) and orthophosphate concentration data in the Passaic River, the Hackensack River, and Newark Bay for over 15 years (2004-2019) were collected along with daily river discharge data from the public database. The annual TIN and orthophosphate (ortho-P) loading from the Passaic River ranged from 915 × 103 kg y-1 to 251 × 104 kg y-1 and 94 × 103 kg y-1to 372 × 103 kg y-1, respectively. The annual TIN and ortho-P loading from the Hackensack River ranged from 3.13 × 103 kg y-1 to 234 × 103 kg y-1 and 0.28 × 103 kg y-1 to 6.97 × 103 kg y-1, respectively. Seasonal variation results indicated that hurricane events highly increased TIN and ortho-P loading from riverine input and reduced residence time in Newark Bay.

Effects of Fermentation on Bioactivity and the Composition of Polyphenols Contained in Polyphenol-Rich Foods: A Review.

Polyphenols, as common components with various functional activities in plants, have become a research hotspot. However, researchers have found that the bioavailability and bioactivity of plant polyphenols is generally low because they are usually in the form of tannins, anthocyanins and glycosides. Polyphenol-rich fermented foods (PFFs) are reported to have better bioavailability and bioactivity than polyphenol-rich foods, because polyphenols are used as substrates during food fermentation and are hydrolyzed into smaller phenolic compounds (such as quercetin, kaempferol, gallic acid, ellagic acid, etc.) with higher bioactivity and bioavailability by polyphenol-associated enzymes (PAEs, e.g., tannases, esterases, phenolic acid decarboxylases and glycosidases). Biotransformation pathways of different polyphenols by PAEs secreted by different microorganisms are different. Meanwhile, polyphenols could also promote the growth of beneficial bacteria during the fermentation process while inhibiting the growth of pathogenic bacteria. Therefore, during the fermentation of PFFs, there must be an interactive relationship between polyphenols and microorganisms. The present study is an integration and analysis of the interaction mechanism between PFFs and microorganisms and is systematically elaborated. The present study will provide some new insights to explore the bioavailability and bioactivity of polyphenol-rich foods and greater exploitation of the availability of functional components (such as polyphenols) in plant-derived foods.

Quantifying global colonization pressures of alien vertebrates from wildlife trade.

The global trade in live wildlife elevates the risk of biological invasions by increasing colonization pressure (the number of alien species introduced to an area). Yet, our understanding of species traded as aliens remains limited. We created a comprehensive global database on live terrestrial vertebrate trade and use it to investigate the number of traded alien species, and correlates of establishment richness for aliens. We identify 7,780 species involved in this trade globally. Approximately 85.7% of these species are traded as aliens, and 12.2% of aliens establish populations. Countries with greater trading power, higher incomes, and larger human populations import more alien species. These countries, along with island nations, emerge as hotspots for establishment richness of aliens. Colonization pressure and insularity consistently promote establishment richness across countries, while socio-economic factors impact specific taxa. Governments must prioritize policies to mitigate the release or escape of traded animals and protect global biosecurity.

Convolutional neural network-based segmentation network applied to image recognition of angiodysplasias lesion under capsule endoscopy.

BACKGROUND: Small intestinal vascular malformations (angiodysplasias) are common causes of small intestinal bleeding. While capsule endoscopy has become the primary diagnostic method for angiodysplasia, manual reading of the entire gastrointestinal tract is time-consuming and requires a heavy workload, which affects the accuracy of diagnosis. AIM: To evaluate whether artificial intelligence can assist the diagnosis and increase the detection rate of angiodysplasias in the small intestine, achieve automatic disease detection, and shorten the capsule endoscopy (CE) reading time. METHODS: A convolutional neural network semantic segmentation model with a feature fusion method, which automatically recognizes the category of vascular dysplasia under CE and draws the lesion contour, thus improving the efficiency and accuracy of identifying small intestinal vascular malformation lesions, was proposed. Resnet-50 was used as the skeleton network to design the fusion mechanism, fuse the shallow and depth features, and classify the images at the pixel level to achieve the segmentation and recognition of vascular dysplasia. The training set and test set were constructed and compared with PSPNet, Deeplab3+, and UperNet. RESULTS: The test set constructed in the study achieved satisfactory results, where pixel accuracy was 99%, mean intersection over union was 0.69, negative predictive value was 98.74%, and positive predictive value was 94.27%. The model parameter was 46.38 M, the float calculation was 467.2 G, and the time length to segment and recognize a picture was 0.6 s. CONCLUSION: Constructing a segmentation network based on deep learning to segment and recognize angiodysplasias lesions is an effective and feasible method for diagnosing angiodysplasias lesions.

Generation of functional human T cell development in NOD/SCID/IL2rγnull humanized mice without using fetal tissue: Application as a model of HIV infection and persistence.

Humanization of mice with functional T cells currently relies on co-implantation of hematopoietic stem cells from fetal liver and autologous fetal thymic tissue (so-called BLT mouse model). Here, we show that NOD/SCID/IL2rγnull mice humanized with cord blood- derived CD34+ cells and implanted with allogeneic pediatric thymic tissues excised during cardiac surgeries (CCST) represent an alternative to BLT mice. CCST mice displayed a strong immune reconstitution, with functional T cells originating from CD34+ progenitor cells. They were equally susceptible to mucosal or intraperitoneal HIV infection and had significantly higher HIV-specific T cell responses. Antiretroviral therapy (ART) robustly suppressed viremia and reduced the frequencies of cells carrying integrated HIV DNA. As in BLT mice, we observed a complete viral rebound following ART interruption, suggesting the presence of HIV reservoirs. In conclusion, CCST mice represent a practical alternative to BLT mice, broadening the use of humanized mice for research.

[Strengths and Difficulties Questionnaire in 737 primary and middle school students aged 6 -17 in Changsha].

OBJECTIVE: To explore the mental health condition of primary and middle school students in Changsha and to provide reference for future evaluation or intervention. METHODS: Strengths and Difficulties Questionnaire (SDQ) (the edition for parents) was used to investigate 737 primary and middle school students aged 6-17 in Changsha. RESULTS: The prevalence of difficulty was 14.11%. The males showed higher scores of problem, hyperactivity and impact, while the females scored higher in emotional symptom as well as prosocial behaviors. Students aged 11-13 and 14-17 showed higher scores of total difficulties, peer problems and emotional symptom than those aged 6-10. Apart from higher scores of total difficulties and peer problems, the other scores were similar to the normal scores in China. there was significant difference in the normal scores between china and other countries. CONCLUSION: mental health problems have divergent characters in gender and age. This study also highlights the importance of establishing local norms of SDQ and the need for appropriate measures in practical situations.