Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

The genetic variation and phylogenetic relationships of Citrus tristeza virus （CTV） isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions （p23, p20, p13, p18, p25, p27, POL, HEL and k17） with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3′ proximal region was relatively conserved, and the 5′ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.

Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences

The coat protein （CP） genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus （CTV） collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations （du） to synonymous mutations （ds） was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV.

KIT and platelet-derived growth factor receptor α wild-type gastrointestinal stromal tumor associated with neurofibromatosis type 1: Two case reports

BACKGROUND Gastrointestinal stromal tumors(GISTs) associated with neurofibromatosis are uncommon compared to their gastrointestinal counterparts. Patients with neurofibromatosis type 1(NF-1) have an increased risk of developing gastrointestinal tumors, including rare types such as GIST.CASE SUMMARY A 60-year-old male Chinese patient was diagnosed with NF-1 10 years ago and presented with upper abdominal discomfort and black stools. Endoscopic ultrasonography and an enhanced abdominal computed tomography scan revealed a mass located 4 cm from the muscular layer of the descending duodenum. A 59-year-old Chinese woman who was diagnosed with NF-1 25 years ago presented with sudden unconsciousness and black stools. Multiple masses in the duodenum were noted by echogastroscopy and an enhanced abdominal computed tomography scan. Both patients presented with cutaneous neurofibromas. The histologic examination of tumors from both patients revealed spindle cells and low mitotic activity. Immunohistochemically, the tumor cells showed strong positivity for KIT(CD117), DOG-1, CD34, and Dehydrogenase Complex Subunit B, and negativity for SMA, desmin, S-100, and β-catenin. None of the six tumors from two patients had KIT exon 9, 11, 13, or 17 or platelet-derived growth factor receptor α exon 12 or 18 mutation, which is a typical finding for sporadic GISTs. None of the six tumors from the two patients had a BRAFV600 E mutation. The patients were alive and well during the follow-up period(range:0.6-5 yr).CONCLUSION There have been only a few previous reports of GISTs associated with NF-1.Although GISTs associated with NF-1 have morphologic and immunohistochemical similarities with GISTs, the pathogenesis, incidence,genetic background, and prognosis are not completely known. A medical history of NF-1 in a patient who has gastrointestinal bleeding or anemia and an intraabdominal mass with nonspecific computed tomography features may help in diagnosing GIST by virtue of the well-known association of these two entities.Molecular genetic studies of cases indicated that GISTs in NF-1 patients have a different pathogenesis than sporadic GISTs.

Changes in gene-expression profiles of colon carcinoma cells induced by wild type K-ras2

AIM: To further elucidate the possible molecular biological activity of wild type K-ras2 gene by detecting changes in wild type K-ras2 gene-induced gene-expression profiles of colon carcinoma cells using cDNA microarray techniques. METHODS: Total RNA was isolated from peripheral blood of health volunteers. Reverse transcription of RNA and polymerase chain reaction were used to synthesize wild type K-ras2 cDNA. K-ras2 cDNA fragment was cloned into a T easy vector and sequenced. A eukaryotic expression vector pCI-neo-K-ras2 was constructed and transfected to Caco2 cell line using the liposome method. Finally, mRNA was isolated, reverse-transcribed to cDNA from pCI-neo-K-ras2 or pCI-neo blank vector-transfected Caco cells, and analyzed by cDNA microarray assay. RESULTS: Restriction enzyme analysis and DNA sequencing verified that the constructed expression vector was accurate. High-quality RNA was extracted and reverse transcribed to cDNA for microarray assay. Among the 135 genes, the expression was up-regulated in 24 and down-regulated in 121. All these differentially expressed genes were related to cell proliferation, differentiation, apoptosis and signal transduction. CONCLUSION: Differentially expressed genes can be successfully screened from wild type K-ras2-transfected colon carcinoma cells using microarray techniques. The results of our study suggest that wild type K-ras2 is related to the negative regulation of cell proliferation, metabolism and transcriptional control, and provide new clues to the further elucidation of its possible biological activity.

Adaptive Response of Wild and Mutant Type Synechococcus cedrorum to a Polychlorinated Pesticide-Endosulfan

The effect of endosulfan, a hexachlorinated pesticide, on growth, inorganic nitrogenous nutrient uptake (NO3-, NO2- and NH4+), change in pigmentation and glycogen content on wild type and chemically mutagenised cells of Synechococcus cedrorum was investigated. The pattern of response to pesticide stress in wild and mutant type was the same. Growth reappeared in both after a period of initial lag in presence of endosulfan. The duration of lag increased with increasing doses of pesticide. Paradoxically, however, the rate of uptake of NO-3, NO-2 and NH+4 pigment and glycogen content progressively increased with increasing doses. The difference in the adaptation response between wild and mutant types was observed only in the concentration of pesticide that could be tolerated; with the mutant tolerating 2.5 fold more.

Therapeutic strategies targeting the epidermal growth factor receptor signaling pathway in metastatic colorectal cancer

More than 1.9 million new colorectal cancer(CRC)cases and 935000 deaths were estimated to occur worldwide in 2020,representing about one in ten cancer cases and deaths.Overall,colorectal ranks third in incidence,but second in mortality.More than half of the patients are in advanced stages at diagnosis.Treatment options are complex because of the heterogeneity of the patient population,including different molecular subtypes.Treatments have included conventional fluorouracil-based chemotherapy,targeted therapy,immunotherapy,etc.In recent years,with the development of genetic testing technology,more and more targeted drugs have been applied to the treatment of CRC,which has further prolonged the survival of metastatic CRC patients.

Successful cetuximab rechallenge in metastatic colorectal cancer:A case report

BACKGROUND Metastatic colorectal cancer(mCRC)treatment has been evolving and increasingly driven by tumor biology and gene expression analysis.Rechallenge with epidermal growth factor receptor(EGFR)inhibitors(anti-EGFR)represents a promising strategy for patients with RAS wild-type(RAS-wt)mCRC and circulating tumor DNA has emerged as a potential selection strategy.Herein,we report the case of a RAS-wt mCRC patient who had a successful response to cetuximab rechallenge.CASE SUMMARY Our patient was diagnosed with stage IV RAS-wt,microsatellite-stable rectosigmoid junction adenocarcinoma.He was started on first-line treatment with FOLFIRI and cetuximab and achieved partial response,allowing for a left hepatectomy(R0),followed by post-operative chemotherapy and an anterior resection;progression-free survival(PFS)of 16 months was obtained.Due to hepatic and nodal relapse,second-line treatment with FOLFOX and bevacizumab was started with partial response;metastasectomy was performed(R0),achieving a PFS of 11 months.After a 15 months anti-EGFR-free interval,FOLFIRI and cetuximab were reintroduced upon disease progression,again with partial response and a PFS of 16 months.Following extensive hepatic relapse,cetuximab was reintroduced and a marked clinical and analytical improvement was seen,after only one cycle.RASwt status was confirmed on circulating tumor DNA.The patient’s overall survival exceeded 5 years.CONCLUSION Our case provides real-world data to support cetuximab rechallenge in later lines of RAS-wt mCRC treatment.

Breeding and Application of a Japonic Rice Cytoplasmic Male Sterility Line, E-Jing A

The Chaling wild abortive type （CL-type） cytoplasmic male sterile line, E-Jing A, was characterized by stable sterility with a sterile plant percentage of 100%, a pollen sterile degree of 98% with dominated typical abortive pollens, and showed the characteristics of good outcrossing with a stigma exsertion rate of 48.2% and an outcrossing rate varied from 30% to 50%, good combining ability, and fine grain quality. E-Jing A was sensitive to temperature. Its duration from sowing to heading was 78-90 d when it was sown from mid May to early June at Wuhan. The breeding procedure of E-Jing A and its characteristics, relationship of restoring and maintaining were introduced.

Peutz-Jeghers syndrome without STK11 mutation may correlate with less severe clinical manifestations in Chinese patients

BACKGROUND Peutz-Jeghers syndrome(PJS) is an autosomal dominant genetic disease with skin mucosal pigment spots and gastrointestinal(GI) multiple hamartoma polyps as clinical characteristics. At present, it is considered that the germline mutation of STK11 gene is the genetic cause of PJS. However, not all PJS patients can be detected STK11 germline mutations. The specific clinical characteristics of these PJS patients without STK11 mutation is an interesting clinical question. Or, like wild type GI stromal tumor, whether these PJS without STK11 mutation are also called PJS is worth discussing. Therefore, we designed the study to understand the clinical characteristics of these PJS patients without STK11 mutation.AIM To investigates whether PJS patients with known STK11 mutations have a more severe spectrum of clinical phenotypes compared to those without.METHODS A total of 92 patients with PJS admitted to the Air Force Medical Center from 2010 to 2022 were randomly selected for study. Genomic DNA samples were extracted from peripheral blood samples, and pathogenic germline mutations of STK11 were detected by high-throughput next-generation gene sequencing. Clinicalpathologic manifestations of patients with and without STK11/LKB1 mutations were compared.RESULTS STK11 germline mutations were observed in 73 patients with PJS. Among 19 patients with no detectable STK11 mutations, six had no pathogenic germline mutations of other genes, while 13 had other genetic mutations. Compared with PJS patients with STK11 mutations, those without tended to be older at the age of initial treatment, age of first intussusception and age of initial surgery. They also had a lower number of total hospitalizations relating to intussusception or intestinal obstruction, and a lower load of small intestine polyps.CONCLUSION PJS patients without STK11 mutations might have less severe clinical-pathologic manifestations than those with.

S14G-humanin restored cellular homeostasis disturbed by amyloid-beta protein

Humanin is a potential therapeutic agent for Alzheimer’s disease, and its derivative, S14G-humanin, is 1 000-fold stronger in its neuroprotective effect against Alzheimer’s disease-relevant insults. Alt-hough effective, the detailed molecular mechanism through which S14G-humanin exerts its effects remains unclear. Data from this study showed that fibril ar amyloid-beta 40 disturbed cel ular ho-meostasis through the cel membrane, increasing intracel ular calcium, generating reactive oxygen species, and decreasing the mitochondrial membrane potential. S14G-humanin restored these re-sponses. The results suggested that S14G-humanin blocked the effects of amyloid-beta 40 on the neuronal cel membrane, and restored the disturbed cel ular homeostasis, thereby exerting a neuroprotective effect on hippocampal neurons.

Signaling interactions among neurons impact cell fitness and death in Alzheimer's disease

The pathology of Alzheimer’s disease involves a long preclinical period,where the characteristic clinical symptoms of the changes in the brain are undetectable.During the preclinical period,homeostatic mechanisms may help prevent widespread cell death.Evidence has pointed towards selective cell death of diseased neurons playing a potentially protective role.As the disease progresses,dysregulation of signaling pathways that govern cell death contributes to neurodegeneration.Aberrant activation of the c-Jun N-terminal kinase pathway has been established in human and animal models of Alzheimer’s disease caused by amyloid-beta 42-or tau-mediated neurodegeneration.Clonal mosaic studies in Drosophila that examine amyloid-beta 42 in a subset of neurons suggest complex interplay between amyloid-beta 42-expressing and wild-type cells.This review examines the role of c-Jun N-terminal kinase signaling in the context of cell competition and short-range signaling interactions between amyloid-beta 42-expressing and wild-type neurons.Cell competition is a conserved phenomenon regulating tissue integrity by assessing the fitness of cells relative to their neighbors and eliminating suboptimal cells.Somatic clones of amyloid-beta 42 that juxtapose genetically distinct neuronal cell populations show promise for studying neurodegeneration.Generating genetic mosaics with labeled clones of amyloid-beta 42-or tau-expressing and wild-type neurons will allow us to understand how short-range signaling alterations trigger cell death in neurons and thereby contribute to the progression of Alzheimer’s disease.These approaches have the potential to uncover biomarkers for early Alzheimer’s disease detection and new therapeutic targets for intervention.

Cultivated Characters of Nandina domestica

[Objective] This study was to systematically investigate the cultivated characters of Nandina domestica and to lay a foundatiQn for breeding, cultivating, as well as its application in landscape. [ Method] Using N. domestica populations from 13 different areas of Hunan Province as tested samples, the cultivated characters of N. domestica in both the cultivated type and wild type were systematically studied through field experiment and comparative analysis. [Resul ] The phenological features of N. domestica from different areas and germplasm resources were basically identical; the growth patterns of cultivated type and wild type were also basically identical, but the cultivated type grew faster than the wild type; both the two types grew quickly at young stage, and either the annual growth of new shoots or the growth pattern of leaves confirmed the fitting curve of the Logistic equation. [ Conclusion ] This research provided basis for the exploitation and utilization the germplasm resources of IV. domestica.

Investigation of hemostatic effect of Spleeninvigorating,Qi-replenishing and Bloodarresting Formula on simvastatin-induced zebrafish hemorrhage model

Blood-arresting Formula on simvastatin-induced zebrafish hemorrhage model compared with that of the Heat-clearing and Blood-cooling Formula.Methods:AB strain wild type zebrafish were treated with 0.5 mM simvastatin for 24 hours to establish the zebrafish hemorrhage model.Under strictly blind experimental conditions,the zebrafish were then treated with experimental agents in different concentration within the maximum non-lethal dose.The intervention effect of the Spleen-invigorating,Qi-replenishing and Blood-arresting Formula was comprehensively assessed by examining the main observational parameters such as the bleeding reduction rate and hemostatic rate while referring to the additional parameters such as blood flow,improvement rate of blood flow,velocity of movement,improvement rate of motion under spleen-qi deficiency.Results:Hemostatic effect:The bleeding rate and hemostatic rate in the zebrafish were respectively,30%,15%,45%,40%and 60%,80%,40%,47%when equal concentration of 500 mg/mL,1000 mg/mL were used.This showed that the experimental herbs B1 was superior to B2 in decreasing the bleeding rate and improving hemostatic effect in the zebrafish.Improvement of blood flow:With equal concentration,the experimental herb B1 was superior to B2 in increasing the blood flow and improvement rate of blood flow in hemorrhagic zebrafish.Promotion of the improvement of motion:With equal concentration of 500 mg/mL and 1000 mg/mL,the experimental herb B1 was superior to B2 in promoting the velocity of motion in the zebrafish and promoting the improvement rate of motion.Conclusion:The manifestations of simvastatin-induced zebrafish hemorrhage are basically similar to that of failure of the spleen to control blood in human beings.The Spleeninvigorating,Qi-replenishing and Blood-arresting Formula has good hemostatic effect on simvastatin-induced hemorrhage in the zebrafish.It also boasts the functions of improving blood flow and velocity of motion in the hemorrhagic zebrafish.Such a conclusion provides the experimental basis for the treatment of the syndrome of failure of the spleen to control blood by this formula.

Induction of Silencing Effect of Swedish Mutant Amyloid Precursor Protein by RNA interference

Summary： Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs （siRNAs） targeted wild type APP （APPwt） and Swedish mutant APP （APPswe）. One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein （GFP）. It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.

Quantitative determination of AI-2 quorum-sensing signal of bacteria using high performance liquid chromatography–tandem mass spectrometry

Autoinducer 2（AI-2）, an important bioactive by-product of the Lux S-catalyzed S-ribosylhomocysteine cleavage reaction in the activated-methyl-cycle, has been suggested to serve as a universal intra- and inter-species signaling molecule. The development of reliable and sensitive methods for quantitative determination of AI-2 is highly desired.However, the chemical properties of AI-2 cause difficulty in its quantitative analysis.Herein, we report a high performance liquid chromatography-tandem mass spectrometric method that enables reproducible and sensitive measurement of AI-2 concentrations in complex matrixes. 4,5-Dimethylbenzene-1,2-diamine（DMBDM）, an easy-to-obtain commercial reagent, was used for the derivatization treatment. The assay was linear in the concentration range of 1.0–1000 ng/m L（R^2= 0.999） and had a lower limit of quantification of0.58 ng/m L. The method exhibited several advantages, e.g., high selectivity, wide linear response range, and good sensitivity. Furthermore, the effectiveness of the method was further validated through measuring AI-2 concentrations in the cell-free culture supernatant from Escherichia coli wild type.

HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis

Deletion of HAX1 in mice causes a severe reduction in the numbers of lymphocytes in the bone marrow and in the spleen. Additionally, B220＋ B progenitor cells in the bone marrow are reduced, suggesting an important function of HAX1 in B cell development. HAX1 is thought to play a protective role in apoptotic processes; therefore, we investigated the role of HAX1 in bone marrow B progenitor cells and splenic B cells. We did not observe an effect on the survival of Hax1-/- bone marrow cells but detected enhanced survival of splenic Hax1-/- B cells upon in vitro starvation/ growth-factor withdrawal. To explain this apparent inconsistency with previous reports of HAX1 function, we also studied the B cell receptor （BCR）-induced apoptosis of IgM-stimulated splenic naive B cells and found that apoptosis decreased in these cells. We further found impaired internalization of the BCR from Hax1-/- splenic B cells after IgM crosslinking; this impaired internalization may result in decreased BCR signaling and, consequently, decreased BCR-mediated apoptosis. We measured HAX1 binding to the cytoplasmic domains of different Ig subtypes and identified KVKWI（V）F as the putative binding motif for HAX1 within the cytoplasmic domains. Because this motif can be found in almost all Ig subtypes, it is likely that HAX1 plays a general role in BCR-mediated internalization events and BCR-mediated apoptosis.

Structural Effects of L16Q, S20G, and L16Q-S20G Mutations on hlAPP： A Comparative Molecular Dynamics Study

The conformation change picture of human islet amyloid polypeptide （hlAPP） is outlined using molecular dynamics simulation, and the structural influences of L16Q, S20G, and L16Q-S20G mutations on the conformation of hlAPP are analyzed. Particularly, the conformational changes of the amyloidogenic-related regions of residues 15-- 17 and 20--29 are emphasized. Our studies find that, for WT hlAPP, residues 15--17 always maintain a stable a-helix structure, residues 20--25 structurally fluctuate between turn and 5-helix, and residues 26--29 mainly adopt coil and bend structures. The hydrogen bonds between the polar groups of hlAPP, long-rang van der Waals forces between the residues, and hydrophobic interactions between the residues of hlAPP are important driving forces to maintain the secondary structure of hlAPP. The replacement of leucine 16 by glutamine stabilizes the helix structure of residues 15--17 and 20--23 of hlAPP monomer, and the structure of residues 24--29 fluctuates be- tween helix and turn. The relatively stable helix structures of residues 15--17 and 20--29 are supposed to be beneficial for L16Q hlAPP to resist the aggregation as observed in the experiment. The substitution of serine20 by glycinc drives residues 15--17 and 20--29 of hlAPP to transform from helix structure to β-strands or coil structures with higher extension and flexibility, which may promote the aggregation of hlAPP as the experiments reported. These results are significant to understand the aggregation mechanism of hlAPP monomer into the dimer, trimer, oligomers and fibrils associated with the type 2 diabetes at the atomic level.

Representation type of local and two-vertex bocses

Let k be an algebraically closed field. It has been proved by Zhang and Xu that if a bocs is of tame representation type, then the degree of the differential of the first solid arrow must be less than or equal to 3. We will prove in the present paper that: The bocs is still wild when the degree of the differential of the first arrow is equal to 3. Especially, the bocs with only one solid arrow is of tame type if and only if the degree of the differential of the arrow is less than or equal to 2. Moreover, we classify in this case the growth problems of the representation category of the bocs and layout the sufficient and necessary conditions when the bocs is of finite representation type, tame domestic and tame exponential growth respectively.