Genetic epidemiology of thalassemia in couples of childbearing age: over 6 years of a thalassemia intervention project.

BACKGROUND: Shenzhen is one of the most populated metropolises in southern China where thalassemia is highly prevalent. The prevention of thalassemia inheritance is an ambition of child-bearing couples. METHODS AND RESULTS: A total of 22,098 peripheral blood samples were collected from 11,049 potentially at-risk couples of childbearing age from Shenzhen. Thalassemia mutations were determined by PCR-based flow-through hybridization. The results identified 45.02% of the participants (9948 out of 22,098) as harboring globin gene mutations, distributed into 18 α-thalassemia alleles detected in 71.48% (7111 out of 9948) and 15 ß-thalassemia alleles detected in 32.68% (3252 out of 9948) of all mutant individuals, among which 415 individuals carried both α- and ß-thalassemia alleles. The most frequent phenotypes for α-globin variations were --SEA/αα (63.37%), -α3.7/αα (18.66%), and -α4.2/αα (7.31%), and those for ß-globin variations were ß41-42/ßN (34.96%), ß654/ßN (28.11%), and ß17/ßN (13.84%). A total of 970 high-risk couples who could possibly give birth to offspring with thalassemia intermedia or major were identified. In addition, the hematological indices were compared among thalassemia genotypes. Significant differences in MCH, MCV, Hb A, and Hb A2 levels among α-thalassemia minor (α+), trait (α0), and intermediate phenotypes (P < 0.05) and between ßE/ßN and the other ß-thalassemia phenotypes (P < 0.05) were found. Moreover, GAP-PCR and next-generation sequencing further identified 42 rare mutations, 13 of which were first reported in the Chinese population. A novel mutation in the ß-globin gene (HBB: c.246 C > A (rs145669504)) was also discovered. CONCLUSIONS: This study presented a comprehensive analysis of thalassemia variations in a population from Shenzhen and may offer valuable insights for thalassemia control and intervention strategies in this area.

Reduction of missed diagnosis of G6PD deficiency in heterozygous females by G6PD/6PGD ratio assay combined with ARMS-PCR.

OBJECTIVE: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that results in impaired enzyme activity. The G6PD/6PGD ratio assay was routinely used for G6PD deficiency screening in China, but there is an apparent defect of missed diagnosis in heterozygous females. The study aims to explore the means to improve its accuracy. METHODS: A total of 4161 Chinese females of childbearing age were collected in this retrospective study. All samples were first subjected to G6PD/6PGD ratio assay and then screened by amplification refractory mutation system PCR (ARMS-PCR) for six hotspot mutants in Chinese population (c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, c.392G>T, and c.871G>A). For the samples with G6PD/6PGD ratio <1.0 and no mutations were found by ARMS-PCR, next-generation sequencing (NGS) was performed. Sanger sequencing was finally used to verify all the variants. RESULTS: The prevalence of G6PD deficiency in Shenzhen females of childbearing age was 7.31%. The proportion of the six hotspot mutations accounted for 98.03% of all 304 G6PD variants carriers. Taking the ARMS-PCR/NGS results as a reference, the missed diagnosis rate of the G6PD/6PGD ratio assay was 33.88%. Using ARMS-PCR to retest the samples with a G6PD/6PGD ratio between 1.00~1.10 or 1.00~1.15 could reduce the missed diagnosis rate from the original 33.88% to 18.09% or 12.05% separately. CONCLUSION: ARMS-PCR is an appropriate supplementary method for discovering most carriers missed by the G6PD/6PGD ratio assay.

Fine mapping and candidate gene analysis of dwarf gene Rht14 in durum wheat (Triticum durum).

Semi-dwarf and dwarf genes were widely used in wheat breeding for improving lodging resistant and increasing yield. Rht14 dwarf gene was identified and deployed in durum wheat, where it showed advantage on important agronomic potential. The reciprocal F2 populations derived of Castelporziano (CP) and Langdon (L) were used for mapping of Rht14, which was located in intervals 4.8 cM and 10.38 cM by KASP (Kompetitive Allele Specific PCR) markers, respectively, where corresponding to 312-454 Mbp on chromosome 6A, and finally, it was mapped to the genomic region of 402 ~ 408 Mbp in Durum Wheat Svevo RefSeq Rel. 1.0 (i.e., 405 ~ 411 Mbp in Chinese Spring RefSeq v.1.0) using recombinants by indel markers. The expression of TdGA2oxA9 was higher in dwarf line than tall lines and the bioactive GA1 was lower. No sequence difference was observed in the promoter and coding region of GA2oxA9 between the dwarf and tall parent, while obvious DNA methylation difference was found in its promoter. Two methylation-related genes with high confidence located in the candidate region and expressed differently between the tall and dwarf ones. This study proposed that Rht14 might regulate the expression of GA2oxA9 by DNA methylation in its promoter, which provided a way to clone Rht14 and to further investigate the mechanism behind.

The fine mapping of dwarf gene Rht5 in bread wheat and its effects on plant height and main agronomic traits.

MAIN CONCLUSION: Rht5 was narrowed to an approximately 1 Mb interval and had pleiotropic effects on plant height, spike length and grain size. TraesCS3B02G025600 was predicted as the possible candidate gene. Plant height is an important component related to plant architecture, lodging resistance, and yield performance. The utilization of dwarf genes has made great contributions to wheat breeding and production. In this study, two F2 populations derived from the crosses of Jinmai47 and Ningchun45 with Marfed M were employed to identify the genetic region of reduce plant height 5 (Rht5), and their derived lines were used to evaluate its effects on plant height and main agronomic traits. Rht5 was fine-mapped between markers Kasp-25 and Kasp-23, in approximately 1 Mb region on chromosome 3BS, which harbored 17 high-confidence annotated genes based on the reference genome of Chinese Spring (IWGSC RefSeq v1.1). TraesCS3B02G025600 were predicted as the possible candidate gene based on its differential expression and sequence variation between dwarf and tall lines and parents. The results of phenotypic evaluation showed that Rht5 had pleiotropic effects on plant height, spike length, culm diameter, grain size and grain yield. The plant height of Rht5 dwarf lines was reduced by an average of 32.67% (32.53 cm) and 27.84% (33.62 cm) in the Jinmai47 and Ningchun45 population, respectively. While Rht5 showed significant and negative pleiotropic effects on culm diameter, aboveground biomass, grain yield, spike length, spikelet number, grain number per spike, grain size, grain weight and filling degree of basal second internode. The culm lodging resistance index (CLRI) of dwarf lines was significantly higher than that of tall lines in the two population. In conclusion, these results lay a foundation for understanding the dwarfing mechanism of Rht5.

Diagnostic value of neutrophil-to-lymphocyte, lymphocyte-to-monocyte and platelet-to-lymphocyte ratio among patients with COVID-19 pneumonia: A retrospective study.

Objectives: Our study was aimed to investigate the clinical characteristics of the patients with COVID-19 pneumonia and research new diagnostic methods for the disease. Methods: In this retrospective study, medical records of 46 novel coronavirus-infected pneumonia (NCIP) patients and 30 healthy individuals in the two multiple hospitals from January 2020 to March 2020 were studied. Clinical characteristics, chest computed tomographic (CT) scans, medicine treatment and laboratory information were collected and retrospectively analyzed. Neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and platelet-to-lymphocyte ratio (PLR) were evaluated. Results: The main symptoms of the patients with NCIP were fever (100%), cough (82.6%), anorexia (37%), expectoration (34.8%) and fatigue (21.7%), dyspnea (15.2%). Ground glass opacity (GGO) with patch shadow was the main observation of the CT imaging (43.4%), followed by GGO (21.7%), patch shadow (19.5%), GGO with consolidation (8.7%) and GGO with reticular pattern (2.1%). The median white blood cell (WBC) count, lymphocyte count, platelet, and lymphocyte-monocyte ratio (LMR) in NCIP group were all significantly lower than in control group (p<0.001, for all comparisons), while the median neutrophil-monocyte ratio (NLR) and platelets-monocyte ratio (PLR) were both significantly higher (p<0.001, for both comparisons). Median WBC count, lymphocyte count, and platelet count on discharge were significantly higher than on admission (p<0.05). Median PLR was significantly lower two weeks after discharge (p<0.001), while NLR remained the same. The area under the curve (AUC) value of WBC, lymphocyte and platelet counts, NLR, LMR and PLR were 0.766, 0.931, 0.655, 0.780, 0.847 and 0.845, respectively. Early stages of the disease were associated with quick changes in WBC, lymphocyte, and platelet levels. However, NLR did not recover even two weeks after the discharge. Conclusion: Changes in WBC, lymphocyte, and platelet counts, as well as NLR, LMR and PLR are strongly associated with COVID-19 pneumonia. Monitoring blood markers may assist in evaluating the progression of the disease.

Lovastatin inhibits the proliferation of human cervical cancer hela cells through the regulation of tp53 pathway by mir-92a-1-5p.

To study the effects of lovastatin on the proliferation, migration, apoptosis and cell cycle of human cervical cancer cell line HeLa in vitro and to investigate the underlying molecular mechanism. The effect of lovastatin on the expression of miRNA in HeLa cells was analyzed using an Affymetrix hybridization chip. The most significantly differentially expressed miRNA and its potential target genes were identified by Ingenuity Pathway Analysis (IPA) and verified by real-time qPCR. Lovastatin could inhibit the proliferation of HeLa cells with the IC50 of 14µmol/L. The proportion of cells in G1 phase increased, while the proportion of cells in S phase and G2/M phase decreased upon treatment with lovastatin. Real-time qPCR found that the expression of miR-192-5p was significantly upregulated (p<0.001) and miR-92a-1-5p was significantly down regulated (p<0.001). Using Ingenuity IPA analysis, MDM2 and TP53 were identified as the potential target genes of miR-192-5p and miR-92a-1-5p, respectively. The expression of TP53 (p<0.01) was significantly up regulated by Lovastatin through miR-92a-1-5p. Lovastatin can inhibit HeLa cell proliferation, promote apoptosis, inhibit migration and block the cell cycle and it may play an anti-tumor effect by down regulating the expression of miR-92a-1-5p and up regulating the expression of TP53.

Purification and characterization of chitinase from Paenibacillus sp. .

The chitinase-producing bacteria Paenibacillus sp. was isolated from soil samples. The chitinase was purified successively by ammonia sulfate fractional precipitation followed by chromatography on DEAE 52-cellulose column and then on Sephadex G-75 column. The chitinase has a molecular weight of ca. 30 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Its optimum pH is 4.5, and its optimum temperature is 50 °C with colloidal chitin as a substrate. The enzyme is stable below 45 °C and in pH ranges between 4.5 and 5.5. It is activated by glucosamine, glucose, N-acetylglucosamine, and metal ions including Ca2+ , Fe2+ , Fe3+ , and Ni2+ . It is inhibited by SDS, H2 O2 , ascorbic acid, Cu2+ , Mg2+ , Ba2+ , Sn2+ , Cr3+ , and K+ . With colloidal chitin as substrate, the Km and the Vmax of the chitinase are 4.28 mg/mL and 14.29 µg/(Min·mL), respectively, whereas the end products of the enzymatic hydrolysis are 14.33% monomer and 85.67% dimer of N-acetylglucosamine. The viscosity of carboxymethyl chitin decreased rapidly at the initial stages when subjected to chitinase hydrolysis, which indicates that the chitinase acts in an endosplitting pattern.

Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in the Shenzhen Population.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. This study aimed to characterize the G6PD gene variant distribution in Shenzhen of Guangdong province. METHODS: A total of 33,562 individuals were selected at the hospital for retrospective analysis, of which 1,213 cases with enzymatic activity-confirmed G6PD deficiency were screened for G6PD gene variants. Amplification refractory mutation system PCR was first used to screen the 6 dominant mutants in the Chinese population (c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, c.392G>T, and c.871G>A). If the 6 hotspot variants were not found, next-generation sequencing was then performed. Finally, Sanger sequencing was used to verify all the mutations. RESULTS: The incidence of G6PD deficiency in this study was 3.54%. A total of 26 kinds of mutants were found in the coding region, except for c.-8-624T>C, which was in the noncoding region. c.1376G>T and c.1388G>A, both located in exon 12, were the top 2 mutants, accounting for 68.43% of all individuals. The 6 hotspot mutations had a cumulative proportion of 94.02%. CONCLUSIONS: This study provided detailed characteristics of G6PD gene variants in Shenzhen, and the results would be valuable to enrich the knowledge of G6PD deficiency.

Double-strand cleavage of DNA by a polyamide-phenazine-di-N-oxide conjugate.

Phenazine and its derivatives have been widely applied as nucleic acid cleavage agents due to active oxygen activating the C-H bond of the substrate. However, diffusion of oxygen radicals limits their potential applications in the DNA-targeted metal-free drug. Introduction of groove binder moiety such as polyamide enhanced the regional stability of radical molecules and reduced cytotoxicity of the drugs. In this work, we described the design and synthesis of a polyamide-modified phenazine-di-N-oxide as a DNA double-strand cleavage agent. The gel assays showed the hybrid conjugates can effectively break DNA double strands in a non-random manner under physiological conditions. The probable binding mode to DNA was investigated by sufficient spectral experiments, revealing weak interaction between hybrid ligand and nucleic acid molecules. The results of our study have implications on the design of groove-binding hybrid molecules as new artificial nucleases and may provide a strategy for developing efficient and safe DNA cleavage reagents.

Succession of bacterial microbiota in tilapia fillets at 4 °C and in situ investigation of spoilers.

The succession of bacterial microbiota in tilapia fillets during cold storage at 4 °C was investigated employing PCR-DGGE method. Results showed that Pseudomonas was the most dominant genus during entire storage period. Shewanella and Psychrobacter were also always present, but became dominant only after 3 days of storage. Acinetobacter, Brevibacterium, Flavobacterium, Dietzia and Janthinobacterium were always the minor genera, among which Acinetobacter and Brevibacterium disappeared 6 days later, and Dietzia and Janthinobacterium only appeared at the end of storage. Further, the potential spoiler(s) of tilapia fillets at 4 °C were investigated in situ. The spoilage ability of a specific group of bacteria was evaluated as follows: Certain preservatives were selectively added to fillets to inhibit a specific group of bacteria, and then the changes in spoilage degree of fillets were determined. In this way the spoilage ability of the inhibited bacteria was evaluated. Our experiments showed that protamine strongly inhibited Pseudomonas but rarely inhibited Psychrobacter, Acinetobacter and Brevibacterium, but garlic juice, on the contrary, strongly inhibited the latter three but rarely inhibited the former. The mixed preservative, which consisted of protamine and garlic juice, didn't play better than protamine alone in preventing the spoilage of fillets. This indicated that Psychrobacter, Acinetobacter and Brevibacterium contribute little to the spoilage of tilapia fillets.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family.

X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease.

Overexpression of COL11A1 confers tamoxifen resistance in breast cancer.

Breast cancer is the most commonly diagnosed malignancy and benefits from endocrine agents such as tamoxifen. However, the development of drug resistance in cancerous cells often leads to recurrence, thus limiting the therapeutic benefit. Identification of potential biomarkers that can predict response to tamoxifen and recognize patients who will clinically benefit from this therapy is urgently needed. In this study, we report that high collagen type XI alpha 1 (COL11A1) expression was associated with poor therapeutic response and prognosis in breast cancer patients treated with tamoxifen. To confirm the role of COL11A1 in the development of tamoxifen resistance, we established MCF-7/COL11A1 and T47D/COL11A1 cell lines, which stably expressed COL11A1. Compared with parental MCF-7 and T47D, MCF-7/COL11A1 and T47D/COL11A1 cells were more resistant to 4-OHT-induced growth inhibition. Moreover, the level of COL11A1 expression was upregulated in tamoxifen-resistant MCF-7/TamR and T47D/TamR cell lines, and depletion of COL11A1 markedly sensitized the cells to 4-OHT in vitro and in vivo. Interestingly, the level of estrogen receptor α (ERα) expression was elevated, probably due to the increased COL11A1 in TamR cells. In addition, knockdown of COL11A1 decreased the expression of ERα and its downstream target genes. Overall, our findings suggest that overexpressed COL11A1 contributes to tamoxifen resistance, and targeting COL11A1 holds great promise for reversing endocrine resistance.

Activities of proteases in deep eutectic solvents and removal of protein from chitin by subtilisin A in betaine/glycerol.

This research intended to remove residual protein from chitin with proteases in deep eutectic solvents (DESs). The activities of some proteases in several DESs, including choline chloride/p-toluenesulfonic acid, betaine/glycerol (Bet/G), choline chloride/malic acid, choline chloride/lactic acid, and choline chloride/urea, which are capable of dissolving chitin, were tested, and only in Bet/G some proteases were found to be active, with subtilisin A, ficin, and bromelain showing higher activity than other proteases. However, the latter two proteases caused degradation of chitin molecules. Further investigation revealed that subtilisin A in Bet/G did not exhibit "pH memory", which is a universal characteristic displayed by enzymes dispersed in organic phases, and the catalytic characteristics of subtilisin A in Bet/G differed significantly from those in aqueous phase. The conditions for protein removal from chitin by subtilisin A in Bet/G were determined: Chitin dissolved in Bet/G with 0.5 % subtilisin A (442.0 U/mg, based on the mass of chitin) was hydrolyzed at 45 °C for 30 min. The residual protein content in chitin decreased from 5.75 % ± 0.10 % to 1.01 % ± 0.12 %, improving protein removal by 57.20 % compared with protein removal obtained by Bet/G alone. The crystallinity and deacetylation degrees of chitin remained unchanged after the treatment.

Novel compound heterozygous MYO15A splicing variants in autosomal recessive non-syndromic hearing loss.

BACKGROUND: Hereditary hearing loss is a highly heterogeneous disorder. This study aimed to identify the genetic cause of a Chinese family with autosomal recessive non-syndromic sensorineural hearing loss (ARNSHL). METHODS: Clinical information and peripheral blood samples were collected from the proband and its parents. Two-step high-throughput next-generation sequencing on the Ion Torrent platform was applied to detect variants as follows. First, long-range PCR was performed to amplify all the regions of the GJB2, GJB3, SLC26A4, and MT-RNR1 genes, followed by next-generation sequencing. If no candidate pathogenetic variants were found, the targeted exon sequencing with AmpliSeq technology was employed to examine another 64 deafness-associated genes. Sanger sequencing was used to identify variants and the lineage co-segregation. The splicing of the MYO15A gene was assessed by in silico bioinformatics prediction and minigene assays. RESULTS: Two candidate MYO15A gene (OMIM, #602,666) heterozygous splicing variants, NG_011634.2 (NM_016239.3): c.6177 + 1G > T and c.9690 + 1G > A, were identified in the proband, and these two variants were both annotated as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Further bioinformatic analysis predicted that the c.6177 + 1G > T variant might cause exon skipping and that the c.9690 + 1G > A variant might activate a cryptic splicing donor site in the downstream intronic region. An in vitro minigene assay confirmed the above predictions. CONCLUSIONS: We identified a compound heterozygous splicing variant in the MYO15A gene in a Han Chinese family with ARNSHL. Our results broaden the spectrum of MYO15A variants, potentially benefiting the early diagnosis, prevention, and treatment of the disease.

A Novel Heterozygous TGFBI c.1613C>A Pathogenic Variant is Associated With Lattice Corneal Dystrophy in a Chinese Family.

PURPOSE: To investigate the gene mutations and relationship of clinical manifestation in a Chinese family with familial lattice corneal dystrophy (LCD). DESIGN: Single-family case-control study. METHODS: A family with familial LCD was recruited for this study. A total of 10 affected and 13 healthy family members participated in this research. Clinical features were examined by slit-lamp examination and anterior segment optical coherence tomography (AS-OCT). Peripheral blood samples were collected from each participant, and genomic DNA was extracted. Whole-exome sequencing (WES) analysis was performed, and the pathogenic variants of LCD were identified using bioinformatics tools and confirmed by Sanger sequencing. RESULTS: Slit-lamp examination revealed diffuse grayish-white punctate, linear, and "lattice-like" opacities in the corneal epithelium and superficial corneal stroma. AS-OCT revealed an irregularly shaped cornea. The corneal epithelium and anterior corneal stroma showed high-reflective deposits and bulges. The clinical appearance of the patients fit the pattern and features of autosomal dominant inheritance of LCD type I (LCD I). A novel pathogenic variant of exon 12 in TGFBI was found by WES analysis, in which cytosine at position 1613 was substituted by adenine (c.1613C>A), and the amino acid was changed from threonine to lysine (p.T538K). Mutated genes and proteins were predicted to be deleterious. CONCLUSION: A novel heterozygous pathogenic variant (c.1613C>A) of TGFBI was identified in the Chinese family with LCD I.

Exploring the Therapeutic Effects of Multifunctional N-Salicylic Acid Tryptamine Derivative against Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Neuroinflammation and oxidative stress play an important role in the whole course of PD, which have been the focus of PD drug development. In our previous research, a series of N-salicylic acid tryptamine derivatives were synthesized, and the biological evaluation showed that the compound LZWL02003 has good anti-neuroinflammatory activity and displayed great therapeutic potency for neurodegenerative disease models. In this work, the neuroprotective efficiency of LZWL02003 against PD in vitro and in vivo has been explored. It was found that LZWL02003 could protect human neuron cells SH-SY5Y from MPP+-induced neuronal damage by inhibiting ROS generation, mitochondrial dysfunction, and cellular apoptosis. Moreover, LZWL02003 could improve cognition, memory, learning, and athletic ability in a rotenone-induced PD rat model. In general, our study has demonstrated that LZWL02003 has good activity against PD in in vitro and in vivo experiments, which can potentially be developed into a therapeutic candidate for PD.

A mouse model for X-linked Alport syndrome induced by Del-ATGG in the Col4a5 gene.

Alport syndrome (AS) is an inherited glomerular basement membrane (GBM) disease leading to end-stage renal disease (ESRD). X-linked AS (XLAS) is caused by pathogenic variants in the COL4A5 gene. Many pathogenic variants causing AS have been detected, but the genetic modifications and pathological alterations leading to ESRD have not been fully characterized. In this study, a novel frameshift variant c.980_983del ATGG in the exon 17 of the COL4A5 gene detected in a patient with XLAS was introduced into a mouse model in by CRISPR/Cas9 system. Through biochemical urinalysis, histopathology, immunofluorescence, and transmission electron microscopy (TEM) detection, the clinical manifestations and pathological alterations of Del-ATGG mice were characterized. From 16 weeks of age, obvious proteinuria was observed and TEM showed typical alterations of XLAS. The pathological changes included glomerular atrophy, increased monocytes in renal interstitial, and the absence of type IV collagen α5. The expression of Col4a5 was significantly decreased in Del-ATGG mouse model. Transcriptomic analysis showed that differentially expressed genes (DEGs) accounted for 17.45% (4,188/24003) of all genes. GO terms indicated that the functions of identified DEGs were associated with cell adhesion, migration, and proliferation, while KEGG terms found enhanced the degradation of ECM, amino acid metabolism, helper T-cell differentiation, various receptor interactions, and several important pathways such as chemokine signaling pathway, NF-kappa B signaling pathway, JAK-STAT signaling pathway. In conclusion, a mouse model with a frameshift variant in the Col4a5 gene has been generated to demonstrate the biochemical, histological, and pathogenic alterations related to AS. Further gene expression profiling and transcriptomic analysis revealed DEGs and enriched pathways potentially related to the disease progression of AS. This Del-ATGG mouse model could be used to further define the genetic modifiers and potential therapeutic targets for XLAS treatment.

JIP3 mediates TrkB axonal anterograde transport and enhances BDNF signaling by directly bridging TrkB with kinesin-1.

Brain-derived neurotrophic factor (BDNF), secreted from target tissues, binds and activates TrkB receptors, located on axonal terminals of the innervating neurons, and thereby initiates retrograde signaling. Long-range anterograde transport of TrkB in axons and dendrites requires kinesin-mediated transport. However, it remains unknown whether anterograde TrkB transport mechanisms are the same in axons versus in dendrites. Here, we show that c-Jun NH(2)-terminal kinase-interacting protein 3 (JIP3) binds directly to TrkB, via a minimal 12 aa domain in the TrkB juxtamembrane region, and links TrkB to kinesin-1. The JIP3/TrkB interaction selectively drives TrkB anterograde transport in axons but not in dendrites of rat hippocampal neurons. Moreover, we find that TrkB axonal transport mediated by JIP3 could regulate BDNF-induced Erk activation and axonal filopodia formation. Our findings demonstrate a role for JIP3-mediated TrkB anterograde axonal transport in recruiting more TrkB into distal axons and facilitating BDNF-induced retrograde signaling and synapse modulation, which provides a novel mechanism of how the TrkB anterograde transport can be coupled to BDNF signaling in distal axons.

mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex pervasive neurodevelopmental disorder and neuroinflammation may contribute to the pathogenesis of ASD. However, the exact mechanisms of abnormal release of proinflammatory mediators in ASD remain poorly understood. This study reports elevated plasma levels of the proinflammatory chemokine (C-C motif) ligand 5 (CCL5) in children with ASD, suggesting an aberrant inflammatory response appearing in the development of ASD. Mining of the expression data of brain or blood tissue from individuals with ASD reveals that mTOR signaling is aberrantly activated in ASD patients. Our in vitro study shows that suppression of mTOR reduces the gene expression and release of CCL5 from human microglia, supporting that CCL5 expression is regulated by mTOR activity. Furthermore, bacterial lipopolysaccharide (LPS)-induced CCL5 expression can be counteracted by siRNA against NF-κB, suggests a determining role of NF-κB in upregulating CCL5 expression. However, a direct regulatory relationship between the NF-κB element and the mTOR signaling pathway was not observed in rapamycin-treated cells. Our results show that the phosphorylated CREB can be induced to suppress CCL5 expression by outcompeting NF-κB in binding to CREB-binding protein (CREBBP) once the mTOR signaling pathway is inhibited. We propose that the activation of mTOR signaling in ASD may induce the suppression of phosphorylation of CREB, which in turn results in the increased binding of CREBBP to NF-κB, a competitor of phosphorylated CREB to drive expression of CCL5. Our study sheds new light on the inflammatory mechanisms of ASD and paves the way for the development of therapeutic strategy for ASD.

Preparation, characterization, and osteogenic activity mechanism of casein phosphopeptide-calcium chelate.

Casein phosphopeptides (CPPs) are good at calcium-binding and intestinal calcium absorption, but there are few studies on the osteogenic activity of CPPs. In this study, the preparation of casein phosphopeptide calcium chelate (CPP-Ca) was optimized on the basis of previous studies, and its peptide-calcium chelating activity was characterized. Subsequently, the effects of CPP-Ca on the proliferation, differentiation, and mineralization of MC3T3-E1 cells were studied, and the differentiation mechanism of CPP-Ca on MC3T3-E1 cells was further elucidated by RNA sequencing (RNA-seq). The results showed that the calcium chelation rate of CPPs was 23.37%, and the calcium content of CPP-Ca reached 2.64 × 105 mg/kg. The test results of Ultraviolet-Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that carboxyl oxygen and amino nitrogen atoms of CPPs might be chelated with calcium during the chelation. Compared with the control group, the proliferation of MC3T3-E1 cells treated with 250 µg/mL of CPP-Ca increased by 21.65%, 26.43%, and 28.43% at 24, 48, and 72 h, respectively, and the alkaline phosphatase (ALP) activity and mineralized calcium nodules of MC3T3-E1 cells were notably increased by 55% and 72%. RNA-seq results showed that 321 differentially expressed genes (DEGs) were found in MC3T3-E1 cells treated with CPP-Ca, including 121 upregulated and 200 downregulated genes. Gene ontology (GO) revealed that the DEGs mainly played important roles in the regulation of cellular components. The enrichment of the Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathway indicated that the AMPK, PI3K-Akt, MAPK, and Wnt signaling pathways were involved in the differentiation of MC3T3-E1 cells. The results of a quantitative real-time PCR (qRT-PCR) showed that compared with the blank control group, the mRNA expressions of Apolipoprotein D (APOD), Osteoglycin (OGN), and Insulin-like growth factor (IGF1) were significantly increased by 2.6, 2.0 and 3.0 times, respectively, while the mRNA levels of NOTUM, WIF1, and LRP4 notably decreased to 2.3, 2.1, and 4.2 times, respectively, which were consistent both in GO functional and KEGG enrichment pathway analysis. This study provided a theoretical basis for CPP-Ca as a nutritional additive in the treatment and prevention of osteoporosis.