A Patient with Partial 17α-Hydroxylase Deficiency Initially Diagnosed with Asherman Syndrome and Pheochromocytoma.

This study present a case of a 49-year-old woman who suffered from resistant hypertension, hypokalemia, hypomenorrhea, and infertility. She was hospitalized 6 years earlier for hypomenorrhea and abdominal pain at the Xiamen Maternity and Child Health Hospital, where she was diagnosed with Asherman syndrome. During hospitalization, a computed tomography examination revealed an adrenal mass. She was referred to Xiamen University Affiliated Zhongshan Hospital for pheochromocytoma and underwent surgical resection of the left adrenal gland. The adrenal cortex adenoma was confirmed by pathological biopsy. Six years later, the patient also presented with hypertension and hypokalemia to our emergency department. A diagnosis of 17α-hydroxylase deficiency was established through the analysis of clinical and laboratory characteristics. The genetic analysis of CYP17A1 revealed compound heterozygous mutations, 1 of which was a mutation of c.1226 C>G, and the other c.297+2T>C.

Cdk5rap3 is essential for intestinal Paneth cell development and maintenance.

Intestinal Paneth cells are professional exocrine cells that play crucial roles in maintenance of homeostatic microbiome, modulation of mucosal immunity, and support for stem cell self-renewal. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Cdk5 activator binding protein Cdk5rap3 (also known as C53 and LZAP) was originally identified as a binding protein of Cdk5 activator p35. Although previous studies have indicated its involvement in a wide range of signaling pathways, the physiological function of Cdk5rap3 remains largely undefined. In this study, we found that Cdk5rap3 deficiency resulted in very early embryonic lethality, indicating its indispensable role in embryogenesis. To further investigate its function in the adult tissues and organs, we generated intestinal epithelial cell (IEC)-specific knockout mouse model to examine its role in intestinal development and tissue homeostasis. IEC-specific deletion of Cdk5rap3 led to nearly complete loss of Paneth cells and increased susceptibility to experimentally induced colitis. Interestingly, Cdk5rap3 deficiency resulted in downregulation of key transcription factors Gfi1 and Sox9, indicating its crucial role in Paneth cell fate specification. Furthermore, Cdk5rap3 is highly expressed in mature Paneth cells. Paneth cell-specific knockout of Cdk5rap3 caused partial loss of Paneth cells, while inducible acute deletion of Cdk5rap3 resulted in disassembly of the rough endoplasmic reticulum (RER) and abnormal zymogen granules in the mature Paneth cells, as well as loss of Paneth cells. Together, our results provide definitive evidence for the essential role of Cdk5rap3 in Paneth cell development and maintenance.

Oral delivery of insulin with intelligent glucose-responsive switch for blood glucose regulation.

BACKGROUND: The traditional treatment for diabetes usually requires frequent insulin injections to maintain normoglycemia, which is painful and difficult to achieve blood glucose control. RESULTS: To solve these problems, a non-invasive and painless oral delivery nanoparticle system with bioadhesive ability was developed by amphipathic 2-nitroimidazole-L-cysteine-alginate (NI-CYS-ALG) conjugates. Moreover, in order to enhance blood glucose regulation, an intelligent glucose-responsive switch in this nanoparticle system was achieved by loading with insulin and glucose oxidase (GOx) which could supply a stimulus-sensitive turnover strategy. In vitro tests illustrated that the insulin release behavior was switched "ON" in response to hyperglycemic state by GOx catalysis and "OFF" by normal glucose levels. Moreover, in vivo tests on type I diabetic rats, this system displayed a significant hypoglycemic effect, avoiding hyperglycemia and maintaining a normal range for up to 14 h after oral administration. CONCLUSION: The stimulus-sensitive turnover strategy with bioadhesive oral delivery mode indicates a potential for the development of synthetic GR-NPs for diabetes therapy, which may provide a rational design of proteins, low molecular drugs, as well as nucleic acids, for intelligent releasing via the oral route.

Bioactive nanoparticle embedded microcapsules for improving the efficacy of type I diabetes therapy.

In order to overcome the side effects of pancreatic transplantation and insulin injection treatment for type I diabetes, we established a drug delivery system employing nanoparticle embedded microcapsules (NEMs). The system co-encapsulated chitosan nanoparticles with γ-aminobutyric acid and ß-TC-6 cells for combined drug and cell therapy in diabetes mellitus (DM). The NEMs, which were formed via high-voltage electrostatic method, had an excellent sphericity with a smooth surface. The average size NEM was 245.52 ± 22.00 µm, which indicated a good size for cell encapsulation. Haemolysis rate of NEMs at concentrations of 100, 200 or 300 mg/mL were all below 5%. Relative viability rates of L929 cells with the same concentrations at 24, 48 or 72 h were all above 80%. We implanted bioactive NEMs into type 1 DM mice to evaluate the effect of the combined therapy. The level of blood glucose in the group receiving the combined therapy decreased during the first 2 weeks of treatment. During the next week, the level of blood glucose stayed in a safe range. Body weight continuously increased during the postoperative period after combined therapy group. Oral glucose tolerance test (OGTT) performed after 24 d showed that the level of blood glucose combined therapy reached the maximum peak of 13.04 mmol/L, lower than 16.56 mmol/L for the cell therapy group. This primary study indicated that microencapsulation technology and combined therapy are promising for the treatment of type I diabetes mellitus.

The influence of spatial distribution on add-on therapy of designed Ca-Alg/CS MEMs system.

To improve the efficacy and reduce the systemic toxicity of the diabetes mellitus, herewith, we developed a novel microparticles-embedded microcapsules (MEMs) system, synthesized from calcium alginate/chitosan (Ca-Alg/CS), by emulsion gelation using a high voltage electrostatic droplet generator. In our study, we selected two antidiabetic drugs insulin (INS) and metformin (MET) as model drugs to investigate different spatial distribution appropriate of MEMs system. Characterization based on particle size and morphology, encapsulation efficiency and drug loading, as well as drug delivery properties were carried out on the MEMs system. Typical multi-chamber structure was shown by SEM and the optical spectra. The average diameters of microparticles and Ca-Alg/CS MEMs were 2100 nm and 410 µm, respectively. Insulin and MET were embedded into MEMs via electrostatic reaction according to FT-IR spectra. Moreover, drug loading and encapsulation efficiency of INS were higher than that of MET in this system when drugs were loaded alone or together. More importantly, this system has potential for orderly drug release and well sustained release when MET in the inner and INS in the outer space could be applied as a combination therapy for diabetes. The obtained in vivo experimental data on diabetes rats has shown that the designed MEMs system resulted in a higher hypoglycemic effect within add-on therapy.

The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology.

Pomegranate (Punica granatum L.) has an ancient cultivation history and has become an emerging profitable fruit crop due to its attractive features such as the bright red appearance and the high abundance of medicinally valuable ellagitannin-based compounds in its peel and aril. However, the limited genomic resources have restricted further elucidation of genetics and evolution of these interesting traits. Here, we report a 274-Mb high-quality draft pomegranate genome sequence, which covers approximately 81.5% of the estimated 336-Mb genome, consists of 2177 scaffolds with an N50 size of 1.7 Mb and contains 30 903 genes. Phylogenomic analysis supported that pomegranate belongs to the Lythraceae family rather than the monogeneric Punicaceae family, and comparative analyses showed that pomegranate and Eucalyptus grandis share the paleotetraploidy event. Integrated genomic and transcriptomic analyses provided insights into the molecular mechanisms underlying the biosynthesis of ellagitannin-based compounds, the colour formation in both peels and arils during pomegranate fruit development, and the unique ovule development processes that are characteristic of pomegranate. This genome sequence provides an important resource to expand our understanding of some unique biological processes and to facilitate both comparative biology studies and crop breeding.

Human heart cell proteins interacting with a C-terminally truncated 2A protein of coxsackie B3 virus: identification by the yeast two-hybrid system.

Protein 2A is a non-structural protein of coxsackievirus B3 (CVB3), an important human pathogen that can cause a variety of human diseases. Protein 2A not only participates in viral life cycle, but also regulates host cell functions; however, the underlying mechanisms remain poorly understood. In order to better understand the molecular mechanisms of CVB3 2A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CVB3 2A interactive proteins in the human heart cDNA library. Full-length 2A shows strong transcriptional activity in yeast cells, which interferes with the application of Y2H system; therefore, a series of 2A deletion mutants were constructed. Analysis of transcriptional self-activation revealed that 2A lost its transcriptional activity after truncation of 60 amino acids (aa) at the N-terminus or deletion of 17 aa at the C-terminus. Choosing the 2A mutant with 17 aa deletion at the C-terminus as the bait protein, four interactive cellular proteins were identified, including TIMP4, MYL2, COX7C, and ENO1. These proteins are mostly related to protein degradation and metabolism. Although the interactions detected by the Y2H system should be considered as preliminary results, the finding of proteins translated from a human heart cDNA library that interacts with the CVB3 2A will stimulate experiments testing the reactivity of a translational mixture derived from that library with full-length 2A protein, followed by co-immunoprecipitation studies.

Identification of the interaction of VP1 with GM130 which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans. Although the susceptibility of CVB3-induced acute pancreatitis is age-dependent, the underlying mechanisms remain unclear. Here we identified the host factor Golgi matrix protein 130 (GM130) as a novel target of CVB3 during CVB3-induced acute pancreatitis. The viral protein VP1 interacted with GM130, disrupted GM130-GRASP65 complexes, and caused GM130 degradation, which may lead to disruption of the Golgi ribbon and development of acute pancreatitis in mice. Interestingly, the expression level of GM130 in mouse pancreas was age-dependent, which was nicely correlated with the age-associated susceptibility of CVB3-induced acute pancreatitis. Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells. Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

In vitro interaction between coxsackievirus B3 VP1 protein and human pleckstrin homology domain retinal protein (PHR1).

Coxsackievirus B3 (CVB3) infection causes central nervous system diseases including aseptic meningitis and encephalitis. To understand the mechanism of this virus, a yeast two-hybrid system was used to screen cellular proteins from a human heart cDNA library. The results revealed that the human Pleckstrin Homology Domain Retinal protein (PHR1), a PH domain-containing protein with low expression in the heart and high expression in the brain, interacts with CVB3 VP1, a major structural protein of CVB3. Yeast mating assays and in vitro coimmunoprecipitation verified the interaction between CVB3 VP1 and PHR1. An α-galactosidase assay indicated that of α-galactosidase activity was higher in positive clones than in controls suggesting a strong interaction. Furthermore, assay of deletion mutants defined the minimal region of PHR1 required for its interaction with VP1 as amino acids 95-172 and two regions of VP1 required for its interaction with PHR1 as amino acids 729-767 and 811-859. The results revealed multiple binding sites between PHR1 and CVB3 VP1 and suggested that the strong interaction between these two proteins might play an important role in central nervous system disease in the human brain.

Vulvovaginal candidiasis: species distribution, fluconazole resistance and drug efflux pump gene overexpression.

The increasing incidence of vulvovaginal candidiasis (VVC) and the emergence of fluconazole resistance are an indisputable fact. However, little information is available regarding the correlation between fluconazole resistance in vaginal Candida albicans and the expression of drug efflux pump genes. In this study, we investigated the species distribution, fluconazole susceptibility profiles and the mechanisms of fluconazole resistance in Candida strains. In total, 785 clinical Candida isolates were collected from patients with VVC. C. albicans was the most frequently isolated species(n = 529) followed by C. glabrata (n = 164) and C. krusei (n = 57). Of all Candida isolates, 4.7% were resistant to fluconazole. We randomly selected 18 fluconazole resistant isolates of C. albicans to evaluate the expression of CDR1, CDR2, MDR1 and FLU1 genes. Compared with fluconazole-susceptible C. albicans isolates, CDR1 gene expression displayed 3.16-fold relative increase, which was statistically significant. CDR2, MDR1 and FLU1 overexpression was observed in several fluconazole-resistant C. albicans isolates, but statistical significance was not achieved. These results demonstrate a high frequency of non-albicans species (32.6%); however, C. albicans is the most common Candida species implicated in vaginitis, and this strain displays considerable fluconazole resistance. Meanwhile, our study further indicates that fluconazole resistance in C. albicans may correlate with CDR1 gene overexpression.

Draft genome of the kiwifruit Actinidia chinensis.

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1 Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage.

12-nt insertion in 3' untranslated region leads to attenuation of classic swine fever virus and protects host against lethal challenge.

We report here the discovery of an attenuation mechanism of classic swine fever virus (CSFV) induced by introduction of a continuous 12-nt (CUUUUUUCUUUU) insertion in viral 3' UTR. The 12-nt insertion sequence was first found in one attenuated vaccine strain HCLV (Hog Cholera Lapinized Virus) which did not exist in other CSFV strains. To address the function of the 12-nt insertion in viral replication and attenuation, we constructed and analyzed two chimeras stemmed from a highly virulent strain Shimen either with introduction of the 12-nt insertion in 3' UTR or the replacement of viral 3' UTR by the 3' UTR of HCLV. We found that the two chimeras' maximum titers declined approximately 100-fold than their parental strain Shimen in PK15 cells. An animal experiment showed that the two chimeras were both dramatically attenuated in pigs. All the chimera-infected pigs survived infection and remained clinically normal with the exception of a transient fever while the 100% mortality was observed for the Shimen-infected pigs. In addition, the two chimeras can induce neutralization antibody to completely protect the pigs against lethal challenge with highly virulent CSFV, which was similar to the vaccine strain HCLV. These data demonstrate that the 12-nt insertion in 3' UTR is sufficient for the attenuation of CSFV. Taken together, a novel attenuation mechanism of CSFV is found and may pave a way to further research for new attenuated vaccine.

A novel role of classical swine fever virus E(rns) glycoprotein in counteracting the newcastle disease virus (NDV)-mediated IFN-beta Induction.

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that E(rns) counteracts Newcastle disease virus (NDV)-mediated induction of IFN-beta. For this purpose, E(rns) fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, E(rns)-EGFP was found to prevent IFN-beta promoter-driven luciferase expression and block the induction of IFN-beta promoter mediated by NDV in a dosedependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-beta mRNA in NDV-infected PK15 cells was observed in the presence of E(rns)-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, E(rns)-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-beta. These evidences establish a novel function for CSFV E(rns) glycoprotein in counteraction of the IFN-beta induction pathway.

Expression and functional characterization of classical swine fever virus E(rns) protein.

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with unusual RNase activity. Recently, E(rns) was found to have a new function of counteracting the beta-interferon (IFN-beta) induction pathway. In this study, wildtype ErnsSM and two mutated E(rns) proteins ErnsH297k and ErnsH346k were expressed in insect cells and purified for RNase activity and function analysis. RNase activity assay in vitro demonstrated that only wildtype E(rns) protein had RNase activity. However, both wildtype ErnsSM and the two mutated E(rns)ErnsH297k and ErnsH346k as exogenous proteins had a block effect on Newcastle disease virus (NDV)-mediated IFN-beta promoter induction.