Molecular and phenotypic characteristics of Bardet-Biedl syndrome in Chinese patients.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a type of non-motile ciliopathy. To date, 26 genes have been reported to be associated with BBS. However, BBS is genetically heterogeneous, with significant clinical overlap with other ciliopathies, which complicates diagnosis. Disability and mortality rates are high in BBS patients; therefore, it is urgent to improve our understanding of BBS. Thus, our study aimed to describe the genotypic and phenotypic spectra of BBS in China and to elucidate genotype-phenotype correlations. METHODS: Twenty Chinese patients diagnosed with BBS were enrolled in this study. We compared the phenotypes of Chinese BBS patients in this study with those from other countries to analyze the phenotypic differences across patients worldwide. In addition, genotype-phenotype correlations were described for our cohort. We also summarized all previously reported cases of BBS in Chinese patients (71 patients) and identified common and specific genetic variants in the Chinese population. RESULTS: Twenty-eight variants, of which 10 are novel, in 5 different BBS-associated genes were identified in 20 Chinese BBS patients. By comparing the phenotypes of BBSome-coding genes (BBS2,7,9) with those of chaperonin-coding genes (BBS10,12), we found that patients with mutations in BBS10 and 12 had an earlier age of onset (1.10 Vs. 2.20, p < 0.01) and diagnosis (4.64 Vs. 13.17, p < 0.01), whereas patients with mutations in BBS2, 7, and 9 had a higher body mass index (28.35 Vs. 24.21, p < 0.05) and more vision problems (p < 0.05). Furthermore, in 91 Chinese BBS patients, mutations were predominant in BBS2 (28.89%) and BBS7 (15.56%), and the most frequent variants were in BBS2: c.534 + 1G > T (10/182 alleles) and BBS7: c.1002delT (7/182 alleles), marking a difference from the genotypic spectra of BBS reported abroad. CONCLUSIONS: We recruited 20 Chinese patients with BBS for genetic and phenotypic analyses, and identified common clinical manifestations, pathogenic genes, and variants. We also described the phenotypic differences across patients worldwide and among different BBS-associated genes. This study involved the largest cohort of Chinese patients with BBS, and provides new insights into the distinctive clinical features of specific pathogenic variants.

Complete genome sequence of Agrobacterium pusense Bbcg2-2, a strain isolated from blueberry crown gall in Taiwan.

Agrobacterium pusense Bbcg2-2 is a strain isolated from a crown gall sample of blueberry (Vaccinium corymbosum) cultivar "Flicker" grown in Taiwan. The complete genome sequence of this bacterium consists of a 2,798,342-bp circular chromosome, a 2,140,031-bp linear chromid, and a 386,016-bp circular plasmid.

Autosomal dominant neurodevelopmental disorders associated with KIF1A gene variants in 6 pediatric patients. / å ­ä¾‹KIF1AåŸºå› å˜å¼‚ç›¸å ³å¸¸æŸ“è‰²ä½“æ˜¾æ€§é—ä¼ ç¥žç»å‘è‚²éšœç¢æ‚£å„¿ä¸´åºŠå’Œé—ä¼ å­¦åˆ†æž.

OBJECTIVES: To analyze the clinical and genetic characteristics of children with autosomal dominant neurodevelopmental disorders caused by kinesin family member 1A (KIF1A) gene variation. METHODS: Clinical and genetic testing data of 6 children with KIF1A gene de novo heterozygous variation diagnosed in Shanghai Children's Medical Center Affiliated to Shanghai Jiao Tong University School of Medicine from the year 2018 to 2020 were retrospectively analyzed. Pathogenic variants were identified based on whole exome sequencing, and verified by Sanger sequencing. Moreover, the effect of variants on three-dimensional structure and stability of protein was analyzed by bioinformatics. RESULTS: Among 6 patients there were 4 males and 2 females, and the age of consultation varied from 7 months to 18 years. All cases had varying degrees of motor developmental delay since childhood, and 4 of them had gait abnormalities or fell easily. In addition, 2 children were accompanied by delayed mental development, epilepsy and abnormal eye development. Genetic tests showed that all 6 cases had heterozygous de novo variations of KIF1A gene, including 4 missense mutations c.296C>T (p.T99M), c.761G>A (p.R254Q), c.326G>T (p.G109V), c.745C>G (p.L249V) and one splicing mutation c.798+1G>A, among which the last three variants have not been previously reported. Bioinformatics analysis showed that G109V and L249V may impair their interaction with the neighboring amino acid residues, thereby impacting protein function and reducing protein stability, and were assessed as "likely pathogenic". Meanwhile, c.798+1G>A may damage an alpha helix in the motor domain of the KIF1A protein, and was assessed as "likely pathogenic". CONCLUSIONS: KIF1A-associated neurological diseases are clinically heterogeneous, with motor developmental delay and abnormal gait often being the most common clinical features. The clinical symptoms in T99M carriers are more severe, while those in R254Q carriers are relatively mild.

Nomogram prediction of the 70-gene signature (MammaPrint) binary and quartile categorized risk using medical history, imaging features and clinicopathological data among Chinese breast cancer patients.

BACKGROUND: The 70-gene signature (70-GS, MammaPrint) test has been recommended by the main guidelines to evaluate prognosis and chemotherapy benefit of hormonal receptor positive human epidermal receptor 2 negative (HR + /Her2-) early breast cancer (BC). However, this expensive assay is not always accessible and affordable worldwide. Based on our previous study, we established nomogram models to predict the binary and quartile categorized risk of 70-GS. METHODS: We retrospectively analyzed a consecutive cohort of 150 female patients with HR + /Her2- BC and eligible 70-GS test. Comparison of 40 parameters including the patients' medical history risk factors, imaging features and clinicopathological characteristics was performed between patients with high risk (N = 62) and low risk (N = 88) of 70-GS test, whereas risk calculations from established models including Clinical Treatment Score Post-5 years (CTS5), Immunohistochemistry 3 (IHC3) and Nottingham Prognostic Index (NPI) were also compared between high vs low binary risk of 70-GS and among ultra-high (N = 12), high (N = 50), low (N = 65) and ultra-low (N = 23) quartile categorized risk of 70-GS. The data of 150 patients were randomly split by 4:1 ratio with training set of 120 patients and testing set 30 patients. Univariate analyses and multivariate logistic regression were performed to establish the two nomogram models to predict the the binary and quartile categorized risk of 70-GS. RESULTS: Compared to 70-GS low-risk patients, the high-risk patients had significantly less cardiovascular co-morbidity (p = 0.034), more grade 3 BC (p = 0.006), lower progesterone receptor (PR) positive percentage (p = 0.007), more Ki67 high BC (≥ 20%, p < 0.001) and no significant differences in all the imaging parameters of ultrasound and mammogram. The IHC3 risk and the NPI calculated score significantly correlated with both the binary and quartile categorized 70-GS risk classifications (both p < 0.001). The area under curve (AUC) of receiver-operating curve (ROC) of nomogram for binary risk prediction were 0.826 (C-index 0.903, 0.799-1.000) for training and 0.737 (C-index 0.785, 0.700-0.870) for validation dataset respectively. The AUC of ROC of nomogram for quartile risk prediction was 0.870 (C-index 0.854, 0.746-0.962) for training and 0.592 (C-index 0.769, 0.703-0.835) for testing set. The prediction accuracy of the nomogram for quartile categorized risk groups were 55.0% (likelihood ratio tests, p < 0.001) and 53.3% (p = 0.04) for training and validation, which more than double the baseline probability of 25%. CONCLUSIONS: To our knowledge, we are the first to establish easy-to-use nomograms to predict the individualized binary (high vs low) and the quartile categorized (ultra-high, high, low and ultra-low) risk classification of 70-GS test with fair performance, which might provide information for treatment choice for those who have no access to the 70-GS testing.

E2/ER signaling mediates the meiotic arrest of goat intrafollicular oocytes induced by follicle-stimulating hormone.

The increased production of high-quality oocytes lies at the heart of the search to accelerate the reproduction of high-quality breeding livestock using assisted reproductive technology. Follicle-stimulating hormone (FSH) maintains the arrest of oocyte meiosis during early follicular development in vivo and promotes the synchronous maturation of nucleus and cytoplasm to improve oocyte quality. However, the mechanism by which FSH maintains meiotic arrest in oocytes is still not fully understood. Oocytes spontaneously resume meiosis once released from the arrested state. In this study, we isolated goat antral follicles with a diameter of 2.0-4.0 mm, cultured them in vitro either with or without added FSH, and finally collected the oocytes to observe their meiotic state. The results showed that FSH effectively inhibited the meiotic recovery of oocytes in follicles [4 h: control (n = 84) vs. with FSH (n = 86), P = .0115; 6 h: control (n = 86) vs. FSH (n = 85), P = 0.0308; and 8 h: control (n = 95) vs. FSH (n = 101), P = 0.0039]. FSH significantly inhibited the downregulation of natriuretic peptide receptor 2 (NPR2) expression and cyclic guanosine monophosphate (cGMP) synthesis during follicular culture in vitro (P < 0.05). Further exploration found that FSH promoted the synthesis of 17ß-estradiol (E2) (P = .0249 at 4 h and P = .0039 at 8 h) and maintained the expression of the estrogen nuclear receptor ERß, but not the estrogen nuclear receptor ERα during follicle culture in vitro (P = .0190 at 2 h, and P = .0100 at 4 h). In addition, E2/ER (estrogen nuclear receptors ERα and ERß) mediated the inhibitory effect of FSH on the downregulation of NPR2 expression and cGMP synthesis, ultimately preventing the meiotic recovery of oocytes (P < .05). In summary, our study showed that FSH-induced estrogen production in goat follicles, and the E2/ER signaling pathway, both mediated meiotic arrest in FSH-induced goat oocytes.

Obtaining a greater number of high-quality oocytes to accelerate the reproduction of high-quality breeding livestock using artificial-assisted reproductive technology remains a pressing problem in animal husbandry and requires further research into the mechanism of oocyte maturation. We investigated the regulatory action of follicle-stimulating hormone (FSH) on the meiosis of oocytes during goat follicle culture in vitro. We found that FSH promoted 17ß-estradiol (E2) synthesis and that E2/ER (estrogen nuclear receptors ERα and ERß)-mediated FSH regulation of the CNP/NPR2 (C-type natriuretic peptide/natriuretic peptide receptor 2) signaling pathway and oocyte meiosis in goat follicles. This study provided an improved theoretical foundation for the increased production of high-quality oocytes using in vitro culture methods.

Case Report: A 13-year-old adolescent diagnosed as malignant phyllodes tumor combined with rhabdomyosarcoma differentiation.

Phyllodes tumor (PT) is an infrequent type of breast neoplasm, constituting a mere 0.5%-1.5% of the entirety of breast tumors. The malignant phyllodes tumor (MPT) comprises only 15% of all phyllodes tumors, and its transformation into rhabdomyosarcoma (RMS) is exceedingly rare in clinical practice. Given its insensitivity to chemotherapy and radiotherapy, treatment options for MPT patients are limited, leaving complete surgical resection as the only option. Therefore, it is imperative to investigate the effective utilization of the heterogeneous differentiation characteristics of MPT to expand treatment alternatives for these patients. In this case report, we represent a 13-year-old adolescent diagnosed with giant breast MPT with RMS differentiation and pulmonary metastasis. The initial step in the treatment process involved radical surgical resection, followed by the administration of four cycles of VDC/IC chemotherapy, which is widely recognized as the standard chemotherapy for RMS. Regrettably, the delay in initiating chemotherapy resulted in minimal observable changes in the size of the pulmonary metastatic nodule. Additionally, a comprehensive literature review on the characterization of MPT with heterogeneous differentiation was conducted to enhance comprehension of the diagnosis and treatment of this uncommon disease in clinical practice. Meanwhile, this case also reminds the doctors that when we diagnose a patient as MPT, it is crucial to consider its heterogenous nature and promptly initiate adjuvant treatment. By targeting the differentiation element of MPT, it becomes feasible to overcome the previously perceived limitation of surgical intervention as the sole treatment option.

Molecular and phenotypic spectrum of cardio-facio-cutaneous syndrome in Chinese patients.

BACKGROUND: Cardio-facio-cutaneous (CFC) syndrome is a RASopathy subtype that presents with unique craniofacial dysmorphology, congenital heart disease, dermatologic abnormalities, growth retardation, and intellectual disability. This study describes the phenotypic spectrum of CFC in China and its association with CFC syndrome gene variants. RESULTS: Twenty Chinese CFC patients, aged 0.6-9.5 years old, were included in this study and their clinical phenotypic spectrum was compared with that of 186 patients with CFC from non-Chinese ethnicities. All 20 Chinese patients with CFC carried de novo heterozygous BRAF, MAP2K1, and MAP2K2 variants. Two novel variants were detected and consistently predicted to be deleterious using bioinformatic tools. The clinical features of CFC in the Chinese patients included hypertrophic cardiomyopathy (2/20, 10%), pulmonary valve stenosis (2/20, 10%), curly or sparse hair (7/20, 35%), epilepsy (1/20, 5%), and hypotonia (10/20, 50%); these features were less frequently observed in Chinese patients than non-Chinese patients (p < 0.05). In contrast, feeding difficulties (19/20, 95%) were more frequently observed in the Chinese patients. Absent eyebrows and severe short stature were more common in patients with BRAF variants than in those with MAP2K1/2 variants. Facial recognition software was used to recognize most CFC patients using artificial intelligence. CONCLUSION: This study identified novel and common variants in our cohort of 20 Chinese patients with CFC. We uncovered differences in clinical features between Chinese and non-Chinese patients and detected genotype-phenotype correlations among the BRAF and MAP2K1/2 variant subgroups. This is the largest cohort of Chinese CFC patients to our knowledge, providing new insights into a subtype of RASopathy.

A high-field cellular DNP-supported solid-state NMR approach to study proteins with sub-cellular specificity.

Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [13C,15N] labeled ubiquitin. We report that SNAPol-1 passively diffuses and homogenously distributes within whole cells and cell nuclei providing ubiquitin spectra of high sensitivity and remarkably improved spectral resolution. For cell nuclei, physical enrichment facilitates a further 4-fold decrease in measurement time and provides an exclusive structural view of the nuclear ubiquitin pool. Taken together, these advancements enable atomic interrogation of protein conformational plasticity at atomic resolution and with sub-cellular specificity.

A novel heterozygous variant of FOXJ1 in a Chinese female with primary ciliary dyskinesia and hydrocephalus: A case report and literature review.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a type of ciliary dyskinesia that is usually caused by autosomal recessive inheritance and can manifest as recurrent respiratory infections, bronchiectasis, infertility, laterality defects, and chronic otolaryngological disease. Although ependymal cilia, which affect the flow of cerebrospinal fluid in the central nervous system, have much in common with respiratory cilia in terms of structure and function, hydrocephalus is rarely associated with PCD. Recently, variants of Forkhead box J1 (FOXJ1) have been found to cause PCD combined with hydrocephalus in a de novo, autosomal dominant inheritance pattern. METHODS: We performed DNA extraction, whole-exome sequencing (WES) analysis, and mutation analysis of FOXJ1 and analyzed the patient's clinical and genetic data. RESULTS: The patient was a 4-year-old female exhibiting normal growth and development. At 3 years and 2 months of age, the patient experienced hand shaking and weakness in the lower limbs. Cardiac ultrasonography showed a right-sided heart, and cranial magnetic resonance imaging showed obstructive hydrocephalus. The nasal nitric oxide level was 54 nL/min. WES indicated a de novo, heterozygous variant of FOXJ1, c.734-735 ins20. This variant was novel, not included in the Human Gene Mutation and Genome Aggregation Database, and likely pathogenic according to the American College of Medical Genetics and Genomics, causing earlier termination of amino acid translation. The patient underwent a neuroendoscopic third ventriculostomy after the diagnosis of obstructive hydrocephalus. Six months after the operation, the patient's motor deficits had improved. CONCLUSION: This is the first report of a de novo, autosomal dominant pattern of FOXJ1 causing PCD combined with hydrocephalus in China. The patient's clinical symptoms were similar to those previously reported. WES confirmed that a novel variant of FOXJ1 was the cause of the PCD combined with hydrocephalus, expanding the spectrum of the genotypes associated with this condition. Physicians should be aware of the correlation of hydrocephalus and PCD and test for FOXJ1 variants.

[Antidepressant mechanism of Shenling Kaixin Granules based on BDNF/TrkB/CREB pathway].

To investigate the antidepressant mechanism of Shenling Kaixin Granules(SLKX) in treating chronic unpredictable mild stress(CUMS) model rats. Ninety male SD rats were randomly divided into control group, model group, Shugan Jieyu Capsules(110 mg·kg~(-1)) group and SLKX low-(90 mg·kg~(-1)), medium-(180 mg·kg~(-1)), and high-dose(360 mg·kg~(-1)) groups. Depression rat model was replicated by CUMS method. After treatment, the behavioral changes of rats were evaluated by sugar preference, open field, elevated cross maze and forced swimming experiments. The contents of interleukin 1 beta(IL-1ß), tumor necrosis factor α(TNF-α), brain-derived neurotrophic factor(BDNF) and 5-hydroxytryptamine(5-HT) in serum were determined by enzyme linked immunosorbent assay(ELISA), and the activities of superoxide dismutase(SOD) and catalase(CAT) in hippocampal CA1 region were also detected. Pathological changes in hippocampal CA1 region were detected by hematoxylin-eosin(HE) staining, and Western blot was used to determine the expression of nerve growth factor(NGF), BDNF, phospho-tyrosine kinase receptor(p-TrkB)/TrkB, phospho-cAMP-response element binding protein(p-CREB)/CREB, nuclear factor E2 related factor 2(Nrf2), heme oxygenase 1(HO-1), B-cell lymphoma-2(Bcl-2)/Bcl-2 associated X protein(Bax) and caspase-3 in hippocampal CA1 region. RESULTS:: showed that compared with the control group, the model group had decreased sugar preference, reduced number of entries and time spent in the center of open field and shortened total distance of movement, reduced number of entries and proportion of time spent in open arm, and increased number and time of immobility in forced swimming experiment. Additionally, the serum contents of IL-1ß and TNF-α and the expression of caspase-3 were higher, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1 and Bcl-2/Bax, and the Nrf2 nuclear translocation were lower in model group than in control group. Compared with the conditions in model group, the sugar preference, the number of entries and time spent in the center of open, total distance of movement, and the number of entries and proportion of time spent in open arm in treatment groups were increased while the number and time of immobility in forced swimming experiment were decreased; the serum contents of IL-1ß and TNF-α and the expression of caspase-3 were down regulated, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1, Bcl-2/Bax, and Nrf2 nuclear translocation were enhanced. In conclusion, SLKX might regulate the Nrf2 nucleus translocation by activating BDNF/TrkB/CREB pathway, lower oxidative stress damage in hippocampus, inhibit caspase-3 activity, and reduce apoptosis of hippocampal nerve cells, thereby playing an antidepressant role.

[Neuroprotective effect of ginsenoside Re on drosophila model of Parkinson's disease].

This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 µmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 µmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.

Spirocyclopiperazinium salt compound DXL-A-24 improves visceral sensation and gut microbiota in a rat model of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is characterized by visceral pain, impaired intestinal barrier and a disorder of the microbiota. DXL-A-24 has analgesic and anti-inflammatory effects by inhibiting neuropeptides and inflammatory factors. In this study, we used chronic unpredictable mild stress (CUMS) induced IBS model, to assess the action of DXL-A-24 on visceral hypersensitivity, barrier function and microbiota. Visceral sensation was assessed by colorectal distension in a model of IBS. The expressions of substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry and western blot, the contents of diamine oxidase (DAO) and D-lactic acid were detected by ELISA, and 16S rRNA to detect the diversity of gut microbiota. CUMS reduced visceral pain threshold and increased colonic permeability of rats. DXL-A-24 for 28 days inhibited these changes. DXL-A-24 also decreased the expression of SP, CGRP in colon and D-LA, DAO in serum. Besides, DXL-A-24 increased the richness and diversity of intestinal microbiota. In conclusions, DXL-A-24 reduced visceral sensitivity, improved intestinal barrier and regulated gut microbiota in rats with IBS.

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods.

OBJECTIVES: To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. METHODS: Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. RESULTS: The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. CONCLUSIONS: The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Dynamics of SARS-CoV-2 Antibody Responses up to 9 Months Post-Vaccination in Individuals with Previous SARS-CoV-2 Infection Receiving Inactivated Vaccines.

Humoral immunity confers protection against COVID-19. The longevity of antibody responses after receiving an inactivated vaccine in individuals with previous SARS-CoV-2 infection is unclear. Plasma samples were collected from 58 individuals with previous SARS-CoV-2 infection and 25 healthy donors (HDs) who had been vaccinated with an inactivated vaccine. The neutralizing antibodies (NAbs) and S1 domain-specific antibodies against the SARS-CoV-2 wild-type and Omicron strains and nucleoside protein (NP)-specific antibodies were measured using a chemiluminescent immunoassay. Statistical analysis was performed using clinical variables and antibodies at different timepoints after SARS-CoV-2 vaccination. NAbs targeting the wild-type or Omicron strain were detected in individuals with previous SARS-CoV-2 infection at 12 months after infection (wild-type: 81%, geometric mean (GM): 20.3 AU/mL; Omicron: 44%, GM: 9.4 AU/mL), and vaccination provided further enhancement of these antibody levels (wild-type: 98%, GM: 53.3 AU/mL; Omicron: 75%, GM: 27.8 AU/mL, at 3 months after vaccination), which were significantly higher than those in HDs receiving a third dose of inactivated vaccine (wild-type: 85%, GM: 33.6 AU/mL; Omicron: 45%, GM: 11.5 AU/mL). The level of NAbs in individuals with previous infection plateaued 6 months after vaccination, but the NAb levels in HDs declined continuously. NAb levels in individuals with previous infection at 3 months post-vaccination were strongly correlated with those at 6 months post-vaccination, and weakly correlated with those before vaccination. NAb levels declined substantially in most individuals, and the rate of antibody decay was negatively correlated with the neutrophil-to-lymphocyte ratio in the blood at discharge. These results suggest that the inactivated vaccine induced robust and durable NAb responses in individuals with previous infection up to 9 months after vaccination.

Identification of genetic and immune signatures for the recurrence of HER2-positive breast cancer after trastuzumab-based treatment.

PURPOSE: To determine the genetic and immune features associated with the recurrence of human epidermal growth factor receptor2-positive (HER2 +) breast cancer (BC) after trastuzumab-based treatment. METHODS: A retrospective cohort study of 48 patients who received trastuzumab-based treatment was divided into recurrent and non-recurrent groups according to clinical follow-up. Baseline samples from all 48 patients were analyzed for genetic variation, HLA allele type, gene expression, and immune features, which were linked to HER2 + BC recurrence. Statistics included logistic regression models, Kaplan-Meier plots, and Univariate Cox proportional hazards models. RESULTS: Compared with the non-recurrent group, the extracellular matrix-related pathway and 3 Hallmark gene sets were enriched in the recurrent group. The infiltration levels of immature B cells and activated B cells were significantly increased in the non-recurrent group, which correlated remarkably with improved overall survival (OS) in two other published gene expression datasets, including TCGA and METABRIC. In the TCGA cohort (n = 275), activated B cells (HR 0.23, 95%CI 0.13-0.43, p < 0.0001), and immature B cells (HR 0.26, 95%CI 0.12-0.59, p < 0.0001). In the METABRIC cohort (n = 236), activated B cells (HR 0.60, 95%CI 0.43-0.83, p = 0.002), and immature B cells (HR 0.65, 95%CI 0.47-0.91, p = 0.011). Cox regression suggested that immature B cells and activated B cells were protective factors for outcome OS. CONCLUSIONS: Aberrant activation of multiple pathways and low baseline tumor-infiltrating B cells are related to HER2 + BC trastuzumab-based recurrence, which primarily affects the antitumor activity of trastuzumab.

Complex clinical manifestations and new insights in RNA sequencing of children with diabetes and WFS1 variants.

Background: WFS1-related disorders involve a wide range of clinical phenotypes, including diabetes mellitus and neurodegeneration. Inheritance patterns of pathogenic variants of this gene can be autosomal recessive or dominant, and differences in penetrance present challenges for accurate diagnosis and genetic counselling. Methods: Three probands and one elder brother from three families were systematically evaluated and the clinical data of other family members were collected from the medical history. Whole-exome sequencing was performed on the probands, and RNA sequencing was performed on four patients, their parents with WFS1 variants, and four gender- and age-matched children with type 1 diabetes mellitus. Results: There were six patients with diabetes. Dilated cardiomyopathy, a rare manifestation of WFS1-related disease, was identified in one patient, along with MRI findings of brain atrophy at age 7 years and 3 months, the earliest age of discovery we know of. Whole-exome sequencing revealed five pathogenic or likely pathogenic variants in the WFS1 gene, including c.1348dupC (p.His450Profs*93), c.1381A>C (p.Thr461pro), c.1329C>G (p.Ser443Arg), c.2081delA (p.Glu694Glyfs*16), c.1350-1356delinsGCA (p.His450Glnfs*26), of which 3 variants (c.1348dupC, c.2081delA, c.1350-1356delinsGCA) were novel that have not been previously reported. The differentially expressed genes were mainly associated with immune-related pathways according to the Gene Ontology enrichment analysis of the RNA sequencing data. The exon 1 region of HLA-DRB1 in two patients was not transcribed, while the transcription of the region in their parents was normal. Conclusion: This study emphasizes the clinical and genetic heterogeneity in patients, even in the same family with WFS1 variants. MRI evaluation of the brain should be considered when WFS1-related disorder is first diagnosed.

Two patients with KDM3B variants and new presentations of Diets-Jongmans syndrome.

KDM3B is located on chromosome 5q31 and encodes KDM3B, which is involved in histone demethylation and epigenetic regulation. Pathogenic KDM3B variants cause a dominantly inherited disorder presenting with intellectual disability (ID), short stature, and facial dysmorphism, named Diets-Jongmans syndrome. We describe two patients with KDM3B variants presenting with Diets-Jongmans syndrome. Genetic testing was performed because of the clinical data and a lack of a clear diagnosis in both patients. Candidate variants were verified by Sanger sequencing. After KDM3B variants were detected, in silico tools were used to predict the pathogenicity of the missense variants. A minigene assay was performed to evaluate the splicing effects of the c.5070 + 1G > A variant on KDM3B. Patient 1 mainly presented with repetitive upper respiratory tract infection and patient 2 presented with palpitation, shortness of breath, and pitting edema; both had ID. Whole exome sequencing identified variants of KDM3B. Patient 1 had the de novo KDM3B c.5070 + 1G > A variant, whereas patient 2 had the c.2828G > A (p.R943Q) variant. Transcriptional experiments of the splicing variant c.5070 + 1G > A revealed aberrant transcripts leading to truncated protein products. We found two pathogenic variants in KDM3B, one of which is novel. Both patients had additional clinical presentations, and patient 1 had transient neutropenia. KDM3B c.5070 + 1G > A is the first KDM3B splice-site variant and was identified as a germline variant. Neutropenia and cardiomyopathy are newly found presentations of Diets-Jongmans syndrome. Our report enriches our knowledge of the genotypic spectrum of the KDM3B variants and phenotypic diversity of Diets-Jongmans syndrome.

First Report of New Guinea Impatiens (Impatiens hawkeri) Leaf Spot Caused by Alternaria burnsii - A. tomato species complex in Taiwan.

New Guinea impatiens (Impatiens hawkeri) is a common ornamental crop usually planted in pots and planters, flower beds, home gardens, or parks in Taiwan. In June 2021, leaf spots on 87.1% (27/31) of potted I. hawkeri plants on National Chung Hsing University (NCHU) campus were observed. Initially, tiny chlorotic leaf spots were found, which aged into brown to grayish white necrotic spots with reddish-purple margins. The necrotic spots enlarged, coalesced, and formed concentric rings. To isolate the pathogen, diseased leaves were surface-disinfected with 70% ethanol for 15 seconds and blotted dry with a paper towel. Small pieces (~2×6 mm2) of tissues were excised from the junction of the lesions and healthy areas, placed onto 2% water agar, and incubated at 25°C with 12-h photoperiod for three days. Individual hyphal tips growing out of diseased tissues were transferred onto potato dextrose agar (PDA). Three isolates, OM10, OM43, and OM45, were obtained and grown on half-strength PDA at 28°C in the dark for at least two weeks. Conidia of each isolate produced on the half-strength PDA were washed off in sterile water with 0.01% of Tween 20. Pathogenicity tests were performed by spraying leaves of 2- to 3-month-old potted healthy I. hawkeri plants with 5 ml of conidial suspension (1 × 105 conidia/ml) of the three isolates, respectively. Control plants were sprayed with sterile water. There were four plants per treatment and the experiments were conducted twice. Inoculated plants were covered with plastic bags for two days and incubated in a greenhouse with a temperature range of 19 to 31°C. Leaf spots similar to those observed in the field were observed at 7 to 14 days after inoculation in both trials. The same fungus was isolated from inoculated plants, whereas control plants showed no symptoms. Thereafter, the three isolates were subjected to morphological and molecular identification. Colonies were brown to gray in the center and white in the border with abundant aerial mycelia. Conidia were brown, obclavate to ovoid, produced in single or branched chains, one to seven transverse and zero to three longitudinal septa. Conidial size of the three isolates ranged between 11.2 to 43.1 × 6.0 to 12.7 µm (n = 50 for each isolate). Conidiophores of the three isolates were dark-brown, septate, branched or unbranched, and measured 27.0 to 147.65 × 2.71 to 4.54 µm (n = 50 for each isolate). Based on the morphological characteristics, the three isolates were identified as Alternaria spp. (Simmons 2007). For molecular identification, the internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and major allergen Alt-a-1 gene (Alt-a-1) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7cR (Sung et al. 2007), gpd1/gpd2 (Berbee et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequence analyses of isolates OM10 (ITS: GenBank Accession no. OP358436; RPB2: OP377483; GAPDH: OP377468; Alt-a-1: OP377471), OM43 (ITS: OP358437; RPB2: OP377484; GAPDH: OP377469; Alt-a-1: OP377472), and OM45 (ITS: OP358438; RPB2: OP377485; GAPDH: OP377470; Alt-a-1: OP377473) showed 100%, 99.61 to 100%, 99.65%, and 100% identities with a reference strain CBS 107.38 of A. burnsii for ITS (KP124420), RPB2 (KP124889), GAPDH (JQ646305), and Alt-a-1 (KP123967), respectively. They also showed 100%, 99.61 to 100%, 99.65%, and 99.58% identities with an A. tomato strain CBS 103.30 for ITS (KP124445), RPB2 (KP124915), GAPDH (KP124294), and Alt-a-1 (KP123991), respectively. Based on the morphological and sequence characteristics, the pathogen causing New Guinea impatiens leaf spot was identified as a member of the Alternaria burnsii - A. tomato species complex. The diseased plants on NCHU campus were destroyed. There have been no reports of the disease in other landscape areas or nurseries. To our knowledge, this is the first report of A. burnsii - A. tomato species complex causing New Guinea impatiens leaf spot in Taiwan. Since the pathogens in the species complex have been documented causing diseases on several important economic crops and the New Guinea impatiens is widely planted in nurseries and landscapes, the host range and the significance of the pathogen in agro-ecosystem may warrant further investigations.

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods / 法医学杂志

OBJECTIVES@#To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods.@*METHODS@#Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis.@*RESULTS@#The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples.@*CONCLUSIONS@#The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Highly bioresistant, hydrophilic and rigidly linked trityl-nitroxide biradicals for cellular high-field dynamic nuclear polarization.

Cellular dynamic nuclear polarization (DNP) has been an effective means of overcoming the intrinsic sensitivity limitations of solid-state nuclear magnetic resonance (ssNMR) spectroscopy, thus enabling atomic-level biomolecular characterization in native environments. Achieving DNP signal enhancement relies on doping biological preparations with biradical polarizing agents (PAs). Unfortunately, PA performance within cells is often limited by their sensitivity to the reductive nature of the cellular lumen. Herein, we report the synthesis and characterization of a highly bioresistant and hydrophilic PA (StaPol-1) comprising the trityl radical OX063 ligated to a gem-diethyl pyrroline nitroxide via a rigid piperazine linker. EPR experiments in the presence of reducing agents such as ascorbate and in HeLa cell lysates demonstrate the reduction resistance of StaPol-1. High DNP enhancements seen in small molecules, proteins and cell lysates at 18.8 T confirm that StaPol-1 is an excellent PA for DNP ssNMR investigations of biomolecular systems at high magnetic fields in reductive environments.