The pathogenic mechanism of syndactyly type V identified in a Hoxd13Q50R knock-in mice.

Syndactyly type V (SDTY5) is an autosomal dominant extremity malformation characterized by fusion of the fourth and fifth metacarpals. In the previous publication, we first identified a heterozygous missense mutation Q50R in homeobox domain (HD) of HOXD13 in a large Chinese family with SDTY5. In order to substantiate the pathogenicity of the variant and elucidate the underlying pathogenic mechanism causing limb malformation, transcription-activator-like effector nucleases (TALEN) was employed to generate a Hoxd13Q50R mutant mouse. The mutant mice exhibited obvious limb malformations including slight brachydactyly and partial syndactyly between digits 2-4 in the heterozygotes, and severe syndactyly, brachydactyly and polydactyly in homozygotes. Focusing on BMP2 and SHH/GREM1/AER-FGF epithelial mesenchymal (e-m) feedback, a crucial signal pathway for limb development, we found the ectopically expressed Shh, Grem1 and Fgf8 and down-regulated Bmp2 in the embryonic limb bud at E10.5 to E12.5. A transcriptome sequencing analysis was conducted on limb buds (LBs) at E11.5, revealing 31 genes that exhibited notable disparities in mRNA level between the Hoxd13Q50R homozygotes and the wild-type. These genes are known to be involved in various processes such as limb development, cell proliferation, migration, and apoptosis. Our findings indicate that the ectopic expression of Shh and Fgf8, in conjunction with the down-regulation of Bmp2, results in a failure of patterning along both the anterior-posterior and proximal-distal axes, as well as a decrease in interdigital programmed cell death (PCD). This cascade ultimately leads to the development of syndactyly and brachydactyly in heterozygous mice, and severe limb malformations in homozygous mice. These findings suggest that abnormal expression of SHH, FGF8, and BMP2 induced by HOXD13Q50R may be responsible for the manifestation of human SDTY5.

Multiscale Synergistic Gecko-Inspired Adhesive for Stable Adhesion under Varying Preload and Surface Roughness.

Inspired by geckos, fibrillar microstructures hold great promise as controllable and reversible adhesives in the engineering field. However, enhancing the adhesion strength and stability of gecko-inspired adhesives (GIAs) under complex real-world contact conditions, such as rough surfaces and varying force fields, is crucial for its commercialization, yet further research is lacking. Here, we propose a hierarchically designed GIA, which features a silicone foam (SF) backing layer and a film-terminated fibrillar microstructure under a subtle multiscale design. This structure has been proven to have a "multiscale synergistic effect", allowing the material to maintain strong and stable adhesion to surfaces with changing normal pressures and roughness. Specifically, under a high load, the adhesive strength is 2 times more than that of conventional GIA, and it is 1.5 times stronger on rough surfaces compared to conventional GIA. Under high pressure and high surface roughness simultaneously, the adhesive strength is 3.3 times higher compared to conventional GIA. Our research demonstrates that the synergistic effect of multiscale biomimetic adhesion structures is highly effective in enhancing the adhesive strength of GIA under some harsh contact conditions.

First-In-Human Study on Pharmacokinetics, Safety, and Tolerability of Single and Multiple Escalating Doses of PA9159 Nasal Spray, a Highly Potent Glucocorticoid in Healthy Chinese Volunteers.

OBJECTIVE: PA9159 (previously named VSG159) is a structurally novel and highly potent glucocorticoid that plays a role in the late development of autoimmune and inflammatory diseases. The current first-in-human ascending-dose study of the PA9159 nasal spray was conducted in healthy Chinese volunteers to evaluate its pharmacokinetics, safety, and tolerability. In addition, the effects of PA9159 on serum cortisol secretion were investigated. METHODS: This was a double-blinded, randomized, placebo-controlled clinical study that included four single-dose groups in the single ascending dose cohort (SAD) and two multiple-dose groups in the multiple ascending dose cohort (MAD), with dose ranges of 10-80 µg and 20-40 µg, respectively. PA9159 was administered bilaterally via nasal spray once only or once daily for seven days. Pharmacokinetic, safety, and tolerability profiles were evaluated. RESULTS: A total of 60 participants completed the study. PA9159 doses of up to 80 µg in the SAD and up to 40 µg in the MAD were shown to be safe and tolerable. The most common treatment-related AEs were mild and transient local nasal AEs. Morning serum cortisol levels approximately remained unchanged in both the single-dose and multiple-dose groups. PA9159 was quantified in 41.8% (368/880) of the samples in all treatment groups, including 25.2% (105/416) of the SAD and 56.7% (263/464) of the MAD. The majority (>80.0%) of PA9159 plasma concentrations ranged from 0.5 to 2 pg/mL in determined samples. The mean AUC0-t of PA9159 in the SAD was 0.91, 1.39±0.68, 11.40±9.91, and 46.30±25.80 h*pg/mL in the 10 to 80 ug single group. The mean terminal half-life time (t1/2) was 8.43 h and 8.97±2.28 h in 40 ug and 80 ug single group, respectively. The mean AUCss of PA9159 in the MAD was 31.70±7.04, 44.20±20.60 h*pg /mL, and the t1/2 was 16.00±4.18 h, 21.20±10.20 h in the 20 ug and 40 ug multiple groups, respectively. The median Tmax was approximately 6 hours in both the SAD and MAD cohorts. CONCLUSIONS: The PA9159 nasal spray was generally safe and well tolerated, and the effects of PA9159 on serum cortisol levels were limited. The plasma concentration and systemic exposure to PA9159 were very low. These findings support the necessity for further clinical studies on PA9159 nasal spray in patients suffering from allergic rhinitis.

Gene replacement therapy in Bietti crystalline corneoretinal dystrophy: an open-label, single-arm, exploratory trial.

Bietti crystalline corneoretinal dystrophy is an inherited retinal disease caused by mutations in CYP4V2, which results in blindness in the working-age population, and there is currently no available treatment. Here, we report the results of the first-in-human clinical trial (NCT04722107) of gene therapy for Bietti crystalline corneoretinal dystrophy, including 12 participants who were followed up for 180-365 days. This open-label, single-arm exploratory trial aimed to assess the safety and efficacy of a recombinant adeno-associated-virus-serotype-2/8 vector encoding the human CYP4V2 protein (rAAV2/8-hCYP4V2). Participants received a single unilateral subretinal injection of 7.5 × 1010 vector genomes of rAAV2/8-hCYP4V2. Overall, 73 treatment-emergent adverse events were reported, with the majority (98.6%) being of mild or moderate intensity and considered to be procedure- or corticosteroid-related; no treatment-related serious adverse events or local/systemic immune toxicities were observed. Compared with that measured at baseline, 77.8% of the treated eyes showed improvement in best-corrected visual acuity (BCVA) on day 180, with a mean ± standard deviation increase of 9.0 ± 10.8 letters in the 9 eyes analyzed (p = 0.021). By day 365, 80% of the treated eyes showed an increase in BCVA, with a mean increase of 11.0 ± 10.6 letters in the 5 eyes assessed (p = 0.125). Importantly, the patients' improvement observed using multifocal electroretinogram, microperimetry, and Visual Function Questionnaire-25 further supported the beneficial effects of the treatment. We conclude that the favorable safety profile and visual improvements identified in this trial encourage the continued development of rAAV2/8-hCYP4V2 (named ZVS101e).

[Genetic analysis of a fetus with cryptophthalmos due to variants of FREM2 gene].

OBJECTIVE: To explore the genetic etiology of a fetus with cryptophthalmos detected by prenatal ultrasonography. METHODS: A fetus undergoing induced labor at 32nd gestational week due to absence of bilateral eye fissures detected by prenatal ultrasonography in January 2017 was selected as the study subject. Umbilical cord blood sample from the fetus and peripheral blood samples from its parents were collected for the extraction of genomic DNA. Pathogenic variants were screened through whole exome sequencing (WES) and verified by Sanger sequencing. Pathogenicity of candidate variants was verified by bioinformatic analysis and protein structure simulation. Based on the results of genetic testing, prenatal diagnosis was provided to the couple upon their subsequent pregnancy. RESULTS: The couple had four adverse pregnancies previously. The aborted fetus was the fifth, with fused bilateral upper and lower eyelids, poorly developed eyeballs, adhesion of the cornea with the upper eyelid, low-set ears, and abnormal plantar creases, and was diagnosed with cryptophthalmos. WES and Sanger sequencing revealed that the fetus has harbored compound heterozygous variants of the FREM2 gene, namely c.4537G>A (p.D1513N) and c.7292C>T (p.T2431M). Both variants were unreported associated with cryptophthalmos previously. Protein structure simulation showed that they may lead to loss of hydrogen bonds in the protein product. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be likely pathogenic (PM1_Supporting+PM2_Supporting+PM5+PP3+PP4; PM2_Supporting+PM3+PP3+PP4). The mother was performed prenatal diagnosis in her sixth pregnancy based on the variants detected in this family, and delivered a daughter with normal phenotype. CONCLUSION: The FREM2: c.4537G>A and c.7292C>T compound heterozygous variants probably underlay the pathogenesis of cryptophthalmos in this fetus. Above finding has enriched the mutational spectrum of the FREM2 gene.

A Method of Reducing Salt Content in Fermented Soy Sauce Improves Its Flavor and Quality.

Most commercially available soy sauce is fermented by high-salt liquid-state (HS) fermentation, which has an excessive salt content and a long fermentation period. In this study, a new salt-reduced fermentation (SR) soy sauce technology involving multiple strains of bacteria was developed to reduce consumers' salt intake. The SR soy sauce was found to have an amino acid nitrogen content of 8.40 g/L and over 80 kinds of flavor substances, which were significantly higher than those of low-salt solid-state fermented soy sauce and approximately equal to HS soy sauce. Compared with HS soy sauce, the salt content of the SR soy sauce was reduced by 59.2%, achieving the salt reduction goal. The proportion of umami amino acids in SR soy sauce reached 32.0% of the total level, enhancing SR soy sauce's quality. Hence, the new fermentation process can decrease salt content and shorten fermentation time.

A novel extraction-free dual HiFi-LAMP assay for detection of methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

[Generation of skin-derived iPSCs from an Osteogenesis imperfecta patient carrying WNT1c.677C>T mutation].

OBJECTIVE: To obtain skin-derived induced pluripotent stem cells (iPSCs) from an Osteogenesis imperfecta (OI) patient carrying WNT1c.677C>T mutation in order to provide a new cell model for investigating the underlying molecular mechanism and stem cell therapy for OI. METHODS: The pathogenic variant of the patient was identified by Sanger sequencing. With informed consent from the patient, skin tissue was biopsied, and primary skin fibroblasts were cultured. Skin fibroblasts were induced into iPSCs using Sendai virus-mediated non-genomic integration reprogramming method. The iPSC cell lines were characterized for pluripotency, differentiation capacity, and karyotyping assay. RESULTS: The patient was found to carry homozygous missense c.677C>T (p.Ser226Leu) mutation of the WNT1 gene. The established iPSC lines possessed self-renewal and capacity for in vitro differentiation. It also has a diploid karyotype (46,XX). CONCLUSION: A patient-specific WNT1 gene mutation (WNT1c.677C>T) iPSC line was established, which can provide a cell model for the study of OI caused by the mutation.

Celastrol-loaded bovine serum albumin nanoparticles target inflamed neutrophils for improved rheumatoid arthritis therapy.

Inflammatory neutrophils (INEs), motivated by cytokines, continue to migrate into the inflamed joints, driving the development of RA. Hence, inducing apoptosis of INEs to reduce recruitment at inflamed joints is an effective strategy for the treatment of RA. However, simply apoptotic INEs may trigger the release of neutrophil extracellular traps (NETs) and accelerate the inflammatory process. To overcome these drawbacks, an RGD-modified bovine serum albumin (BSA) nanoparticles (CBR NPs) was fabricated to selectively target INEs in situ for intracellular delivery of CLT. Studies have demonstrated that CBR NPs can selectively target circulating INEs and induce INEs apoptosis. Meanwhile, CBR NPs inhibited the activation of NETs via NF-κB pathway and the release of Cit-H3 thereby blocking the release process of NETs. In collagen-induced arthritis (CIA) mouse model, CBR NPs suppressed the inflammatory response, and reduced the toxic effects of CLT. In summary, this study shed light on an innovative approach to treat RA by inducing apoptosis of circulating INEs and inhibiting NETs. STATEMENT OF SIGNIFICANCE: RGD-modified bovine serum albumin (BSA) nanoparticles for delivering celastrol, abbreviated as CBR NPs, were constructed to inhibit the infiltration of circulating inflammatory neutrophils (INEs) into inflamed joints while inhibiting the release of NETs to alleviate tissue damage. CBR NPs were prepared for the first time to induce apoptosis of INEs; CBR NPs could inhibit the release of NETs while inducing apoptosis of INEs in vivo and vitro cellular experiments; CBR NPs had favorable anti-inflammatory effects and low toxicity side-effects in collagen-induced arthritis (CIA) mouse models. The application of nanotechnology to induce apoptosis of INEs while inhibiting the release of NETs was a promising approach for the treatment of RA.

Cognitive flexibility and entrepreneurial creativity: the chain mediating effect of entrepreneurial alertness and entrepreneurial self-efficacy.

Entrepreneurial creativity is an important part of entrepreneurs' competency structure, and studies have been conducted to explore its impact on outcome variables like entrepreneurial performance, etc., but there are fewer studies on its antecedent variables at the individual level. In the relational model of entrepreneurial creativity, cognitive flexibility, entrepreneurial alertness, and entrepreneurial self-efficacy are included to construct a mediated use spss26.0 model, and data are collected from a research sample of 325 entrepreneurs for empirical analysis. The results indicate that: cognitive flexibility has a positive effect on entrepreneurial creativity; entrepreneurial alertness plays an independent mediating role between cognitive flexibility and entrepreneurial creativity, which is similar to how entrepreneurial self-efficacy plays the role between cognitive flexibility and creativity; entrepreneurial alertness and entrepreneurial self-efficacy then play a chain mediating role between cognitive flexibility and creativity. These findings reveal that three variables jointly influence entrepreneurial creativity, providing new theoretical and practical insights for understanding and enhancing entrepreneurial creativity. In addition, the study provides valuable guidance for entrepreneurship education and training, which can help entrepreneurs to better utilize their creativity and thus promote innovation and success in entrepreneurial activities.

An SS31-rapamycin conjugate via RBC hitchhiking for reversing acute kidney injury.

Mitochondrial dysfunction plays a major role in driving acute kidney injury (AKI) via alteration in energy and oxygen supply, which creates further ROS and inflammatory responses. However, mitochondrial targeting medicine in recovering AKI is challenging. Herein, we conjugated SS31, a mitochondria-targeted antioxidant tetrapeptide connecting a cleavable linker to rapamycin (Rapa), which provided specific interaction with FK506-binding protein (FKBP) in the RBCs. Once entering the bloodstream, SS31-Rapa could be directed to the intracellular space of RBCs, allowing the slow diffusion of the conjugate to tissues via the concentration gradient. The new RBC hitchhiking strategy enables the encapsulation of conjugate into RBC via a less traumatic and more natural and permissive manner, resulting in prolonging the t1/2 of SS31 by 6.9 folds. SS31-Rapa underwent the direct cellular uptake, instead of the lysosomal pathway, released SS31 in response to activated caspase-3 stimulation in apoptotic cells, favoring the mitochondrial accumulation of SS31. Combined with autophagy induction associated with Rapa, a single dose of SS31-Rapa can effectively reverse cisplatin and ischemia reperfusion-induced AKI. This work thus highlights a simple and effective RBC hitchhiking strategy and a clinically translatable platform technology to improve the outcome of other mitochondrial dysfunctional related diseases.

Enhanced soy sauce stability and reduced precipitation by improving critical steps in the fermentation process.

Conventional high-salt dilute-state soy sauce is vulnerable to precipitation after processing, which will reduce the systemic stability and nutrition of soy sauce. This work aims to optimize key steps of the soy sauce fermentation process to improve its stability and reduce precipitation. The amino acid nitrogen (AAN) and the total nitrogen (TN) contents of the new soy sauce were 8.3 g/L and 18.7 g/L, which were significantly enhanced by 33.9% and 14.0%, respectively, compared to the control group. More flavor substances were detected in the new soy sauce, including furans and pyrazines, which contribute to the special flavor of soy sauce. The particle size distribution curve was significantly shifted to the left, and the absolute value of zeta-potential increased. The new fermentation process soy sauce had a higher raw material utilization rate, smaller average particle size of 15.56 µm, and significantly higher stability when combined with the rheological examination. Consequently, the quality and flavor of soy sauce can be improved by using the new fermentation process.

pH-Responsive Injectable Multifunctional Pluronic F127/Gelatin-Based Hydrogels with Hydrogen Production for Treating Diabetic Wounds.

Diabetic chronic wounds remain a major clinical challenge with long-term inflammatory responses and extreme oxidative damage. Hence, a pH-responsive injectable multifunctional hydrogel [Gel/CUR-FCHO/Mg (GCM) micromotors] via a Schiff base reaction between gelatin and benzaldehyde-grafted Pluronic F127 drug-loaded micelles (FCHO) was fabricated for the first time. Dynamic Schiff base linkage endowed the GCM hydrogel with the ability to be self-healing, injectable, and pH-responsive for on-demand drug delivery at the wound site. Curcumin (CUR), a hydrophobic drug with antioxidative, anti-inflammatory, and antibacterial activities, was encapsulated into the hydrogel matrix by micellization (CUR-FCHO micelles). Simultaneously, magnesium-based micromotors (Mg micromotors) were physically entrapped into the system for providing active hydrogen (H2) to scavenge reactive oxygen species and alleviate inflammatory responses. As a result, the GCM micromotor hydrogel displayed an inherent antibacterial property, extraordinary antioxidative performance, and remarkable biocompatibility. In the diabetic mouse with a full-thickness cutaneous defect wound, the GCM hydrogel could remodel the inflammatory microenvironment and stimulate vascularization and collagen deposition, thereby facilitating wound closure and enhancing tissue regeneration, which offered a promising therapeutic option for diabetic chronic wound management.

Coffee Leaf Tea Extracts Improve Hyperuricemia Nephropathy and Its Associated Negative Effect in Gut Microbiota and Amino Acid Metabolism in Rats.

Hyperuricemia nephropathy (HN) is a metabolic disease characterized by tubular damage, tubulointerstitial fibrosis, and uric acid kidney stones and has been demonstrated to be associated with hyperuricemia. Coffee leaf tea is drunk as a functional beverage. However, its prevention effects on HN remain to be explored. This study showed that coffee leaf tea extracts (TE) contain 19 polyphenols, with a total content of 550.15 ± 27.58 mg GAE/g. TE decreased serum uric acid levels via inhibiting XOD activities and modulating the expression of urate transporters (GLUT9, OAT3, and ABCG2) in HN rats. TE prevented HN-induced liver and kidney damage and attenuated renal fibrosis. Moreover, it upregulated the abundance of SCFA-producing bacteria (Phascolarctobacterium, Alloprevotella, and Butyricicoccus) in the gut and reversed the amino acid-related metabolism disorder caused by HN. TE also decreased the circulating LPS and d-lactate levels and increased the fecal SCFA levels. This study supported the preliminary and indicative effect of coffee leaf tea in the prevention of hyperuricemia and HN.

Stimuli-responsive nanoparticles delivered by a nasal-brain pathway alleviate depression-like behavior through extensively scavenging ROS.

Depression is one of the most common mental diseases, which seriously affects patients' physical and mental health. Emerging evidence has indicated that oxidative stress (OS) is a major cause of neurodegeneration involved in the pathogenesis of depression. Consequently, targeted reactive oxygen species (ROS) elimination is regarded as a promising strategy for efficient depression therapy. In addition, insufficient brain drug delivery is the main obstacle to depression therapy owing to the presence of the blood-brain barrier (BBB). To achieve the goals of bypassing the BBB and promoting antioxidant therapy for depression, a broad-spectrum ROS scavenging NPs was rationally designed through a nasal-brain pathway developed for combined ROS scavenging and brain drug delivery. A hexa-arginine (R6) modified ROS-responsive dextran (DEX) derivate was synthesized for antidepressant olanzapine (Olz) and H2 donor amino borane (AB) loading to prepare Olz/RDPA nanoparticles (NPs). Subsequently, the NPs were dispersed into a thermoresponsive hydrogel system based on poloxamer. In vitro and in vivo results demonstrated that Olz/RDPA in situ thermoresponsive hydrogel system could effectively deliver NPs to the brain via the nasal-brain pathway and alleviate depression-like behaviors through combined ROS depletion and inhibition of 5-HT dysfunction of the oxidative stress-induced. The proposed ROS-scavenging nanotherapeutic would open a new window for depression treatment. STATEMENT OF SIGNIFICANCE: ROS is an innovative therapeutic target involving the pathology of depression whereas targeted delivery of ROS scavenging has not been achieved yet. In the current study, ROS-responsive nanoparticles (Olz/RDPA NPs) were prepared and dispersed in a thermosensitive hydrogel for delivery through the nasal-brain pathway for the treatment of depression. Sufficient ROS depletion and improvement of delivery capacity by the nasal-brain pathway effectively could reverse oxidative stress and alleviate depressive-like behavior. Collectively, these nanoparticles may represent a promising strategy for the treatment of depression.

[Genetic analysis of two families with Short-rib thoracic dysplasia type 3].

OBJECTIVE: To explore the pathogenic variants and clinical classification of two fetuses with Short-rib thoracic dysplasia with or without polydactyly (SRTD). METHODS: With informed consent obtained, the phenotypic characteristics of the fetuses were comprehensively examined, and genomic DNA was extracted from fetal skin tissue and peripheral blood samples of the parents with conventional phenol-chloroform method. Whole exome sequencing (WES) was carried out on both fetuses, and the candidate variants were validated by Sanger sequencing. The pathogenicity of the candidate variants was analyzed using bioinformatic software VarCards, and the impact of the variants on the protein structure was predicted with Swiss-Pdb-viewer. RESULTS: Both fetuses were found to harbor compound heterozygous variants of the DYNC2H1 gene, including c.515C>A (p.Pro172Gln) and c.5983G>A (p.Ala1995Thr) in fetus 1, and c.5920G>T (pGly1974) and c.9908T>C (p.He3303Thr) in fetus 2. The parents of both fetuses were heterozygous carriers. CONCLUSION: The compound heterozygous variants of the DYNC2H1 gene probably underlay the SRTD3 in the two fetuses.

Identification of potential susceptibility genes in patients with primary Sjögren's syndrome-associated pulmonary arterial hypertension through whole exome sequencing.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare complication of primary Sjögren's syndrome (pSS). Several genes have proven to be associated with pSS and PAH. However, there is no study specifically addressing the genetic susceptibility in pSS combined with PAH. METHODS: Thirty-four unrelated patients with pSS-PAH were recruited from April 2019 to July 2021 at Peking Union Medical College Hospital. Demographic and clinical data were recorded in detail, and peripheral blood samples were collected for whole-exome sequencing (WES). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to predict the functional effect of mutant genes. Genetic variants identified by WES were confirmed by polymerase chain reaction (PCR)-Sanger sequencing. RESULTS: We totally identified 141 pathogenic variant loci of 129 genes in these 34 pSS-PAH patients, using WES analysis. Patients with a family history of rheumatic diseases are more likely to carry FLG mutations or carry gene variations related to the biosynthesis of the amino acids pathway (p < 0.05). According to Sanger sequencing confirmation and pathogenicity validation, we totally identified five candidate pathogenic variants including FLG c.12064A > T, BCR c.3275_3278dupCCGG, GIGYF2 c.3463C > A, ITK c.1741C > T, and SLC26A4 c.919-2A > G. CONCLUSION: Our findings provide preliminary data of exome sequencing to identify susceptibility loci for pSS-PAH and enriched our understanding of the genetic etiology for pSS-PAH. The candidate pathogenic genes may be the potential genetic markers for early warning of this disease.

Clinical and genetic analysis in Chinese families with synpolydactyly, and cellular localization of HOXD13 with different length of polyalanine tract.

Synpolydactyly (SPD) is caused by mutations in the transcription factor gene HOXD13. Such mutations include polyalanine expansion (PAE), but further study is required for the phenotypic spectrum characteristics of HOXD13 PAE. We investigated four unrelated Chinese families with significant limb malformations. Three PAEs were found in the HOXD13 polyalanine coding region: c.172_192dup (p.Ala58_Ala64dup) in Family 1, c.169_192dup (p.Ala57_Ala64dup) in Family 2, and c.183_210dup (p.Ala62_Ala70dup) in Family 3 and Family 4. Interestingly, we identified a new manifestation of preaxial polydactyly in both hands in a pediatric patient with an expansion of seven alanines, a phenotype not previously noted in SPD patients. Comparing with the wild-type cells and mutant cells with polyalanine contractions (PACs), the HOXD13 protein with a PAE of nine-alanine or more was difficult to enter the nucleus, and easy to form inclusion bodies in the cytoplasm, and with the increase of PAE, the more inclusion bodies were formed. This study not only expanded the phenotypic spectrum of SPD, but also enriched our understanding of its pathogenic mechanisms.

CRISPR/Cas9 correction of a dominant cis-double-variant in COL1A1 isolated from a patient with osteogenesis imperfecta increases the osteogenic capacity of induced pluripotent stem cells.

Osteogenesis imperfecta (OI) is a hereditary skeletal disorder that is mainly caused by variants in COL1A1/2. So far, no specific treatment has been developed to correct its underlying etiology. We aimed to gain a better understanding of the pathological mechanisms of OI and develop gene therapies to correct OI-causing variants. A de novel cis-double-variant c.[175C>T; 187T>A] in COL1A1 was identified from a 5-year-old OI patient by whole-exome sequencing (WES). Three peptide nucleic acids (PNAs) were designed and then transfected patient-derived fibroblasts. PNA2 affected the translational strand and induced an optimal interfering effect at 0.25µM concentration, proved by Sanger sequencing, qPCR, Western blot, and immunostaining. Additionally, induced pluripotent stem cells (iPSCs) were cultured from patient-derived fibroblasts. Clones of iPSCs with c.187T>A variant and those with both variants largely restored their osteogenic capacities after CRISPR/Cas9 gene editing, which corrected the variants. Importantly, correcting c.187T>A variant alone in CRISPR-edited iPSCs was sufficient to alleviate OI phenotypes, as indicated by increased levels of COL1A1, COL1A2, ALP mRNAs, and COL1A1 protein. Our findings suggest that c.187T>A is the dominant variant of cis-double-variant in COL1A1 that led to OI, and PNA interference and CRISPR/Cas9 gene editing may be new therapeutic tools for OI treatment. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A Bioequivalence Study With Pharmacokinetic Endpoints for Azithromycin Eye Drops.

Azithromycin eye drops with a bioadhesive ocular drug-delivery system can offer a simplified dosing regimen. In this study, we compared the pharmacokinetic properties and assessed the bioequivalence of a newly developed generic azithromycin eye drop with a branded formulation. This open-label, single-dose, randomized, crossover, sparse-sampling ocular bioequivalence study was conducted on 48 healthy Chinese volunteers. Tear samples were collected for up to 36 hours, and each participant was randomly allocated to one of the prespecified sampling times. Tear drug concentrations were determined using a validated liquid chromatography-tandem mass spectrometry method. The pharmacokinetic parameters were calculated via noncompartmental analysis. A nonparametric bootstrap method was used to obtain 90% confidence intervals (CIs) for the ratios of the test and reference drugs. Tolerability was evaluated for adverse events (AEs). After bootstrapping (1000 iterations), the 90%CIs for the log-transformed ratios of Cmax , AUC0-t , and AUC0-∞ were within the acceptable bioequivalence range (80%-125%). No moderate-to-severe AEs were reported for either formulation. Bioequivalence was demonstrated between the two formulations. The sparse-sampling design with the bootstrapping technique is promising for bioequivalence studies of topical ophthalmic drugs.