[Clinical application of convolutional neural network in pathological diagnosis of metastatic lymph nodes of gastric cancer].

Objective: To examine the value and clinical application of convolutional neural network in pathological diagnosis of metastatic lymph nodes of gastric cancer. Methods: Totally 124 patients with advanced gastric cancer who underwent radical gastrectomy plus D2 lymphadenectomy at Affiliated Hospital of Qingdao University from July 2016 to December 2018 were selected in the study. According to the chronological order, the first 80 cases were served as learning group. The remaining 44 cases were served as verification group. There were 45 males and 35 females in the study group, with average age of 57.6 years. There were 29 males and 15 females in the validation group, with average age of 9.2 years. The pre-training convolutional neural network architecture Resnet50 was trained and fine-tuned by 21 352 patches with cancer areas and 14 997 patches without cancer areas in the training group. A total of 78 whole-slide image served as a test dataset including positive (n=38) and negative (n=40) lymph nodes. The convolutional neural network computer-aided detection (CNN-CAD) system was used to analyze the ability of convolutional neural network system to screen metastatic lymph nodes at the level of slice by setting threshold, and evaluate the system's classification accuracy by calculating its sensitivity, specificity, positive predictive value, negative predictive value and area under the receiver operating characteristic curve (AUC). Results: The classification accuracy of CNN-CAD system at slice level was 100%.The AUC for the CNN-CAD system was 0.89. The sensitivity was 0.778, specificity was 0.995, overall accuracy was 0.989. Positive and negative predictive values were 0.822 and 0.994, respectively. The CNN-CAD system achieved the same classification results as pathologists. Conclusions: The CNN-CAD system has been constructed to distinguished benign and malignant lymph node slides with high accuracy and specificity. It could achieve the similar classification results as pathologists.

Bendamustine added to allogeneic conditioning improves long-term outcomes in patients with CLL.

Bendamustine has shown a favorable safety profile when included in chemotherapy regimens for several types of lymphoma, including CLL. This study investigated the long-term effect of adding bendamustine to a conditioning regimen on survival, rate of engraftment, immune recovery and GvHD after allogeneic stem cell transplantation (alloSCT) in CLL patients. These outcomes were compared with the fludarabine, cyclophosphamide and rituximab (FCR) conditioning regimen. We reviewed the data for 89 CLL patients treated on three trials at our institution. Twenty-six (29%) patients received bendamustine, fludarabine and rituximab (BFR) and 63 (71%) received FCR. Patient characteristics were similar in both groups. Ten (38%) BFR-treated patients vs only two (3%) FCR-treated patients did not experience severe neutropenia (P=<0.001). The 3-year overall survival estimates for the BFR and FCR groups were 82 and 51% (P=0.03), and the 3-year PFS estimates were 63% and 27% (P=0.001), respectively. The 2-year treatment-related mortality was 8 and 23% and the incidence of grade 3 or 4 GvHD was 4% and 10%, respectively. This study is the first to report that addition of bendamustine to alloSCT conditioning for CLL patients is associated with improved survival and lower mortality, myelosuppression, and GvHD.

Ginsenoside Rg3 attenuated omethoate-induced lung injury in rats.

Organophosphorus exposure affects different organs such as the lung, gastrointestinal tract, liver, and brain. The present experiment aimed to evaluate the effect of ginsenoside Rg3 on lung injury induced by acute omethoate poisoning. Rats were administered with omethoate subcutaneously at a single dose of 60 mg/kg, followed by ginsenoside Rg3 (5, 10, or 20 mg/kg) treatment. Histopathological examination of the lung was performed at 24 h after the omethoate exposure. The antioxidative parameters in the lung were also assayed. Moreover, the activities of acetylcholinesterase, myeloperoxidase, and the content of tumor necrosis factor α (TNF-α) in the lung were determined. The results showed that ginsenoside Rg3 attenuated omethoate-induced lung injury. Ginsenoside Rg3 increased the level of glutathione in the lung (p < 0.05 or p < 0.01). The altered activities of superoxide dismutase and catalase in the lung were also ameliorated by ginsenoside Rg3 treatment (p < 0.05 or p < 0.01). Ginsenoside Rg3 caused significant reductions in the contents of malondialdehyde, TNF-α, and the activity of myeloperoxidase (p < 0.05 or p < 0.01). The present study demonstrated that ginsenoside Rg3 had a protective effect against omethoate-induced lung injury in rats, and the mechanisms were related to its antioxidant potential and anti-inflammatory effect.

[Molecular cloning, structural analysis, and expression of zona pellucida glycoprotein ZP3 gene from Chinese zokor, Myospalax fontanierii].

The zona pellucida 3 (ZP3) plays a crucial role in reproductive immunology. We obtained a full-length cDNA encoding Chinese Zokor zp3, using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1269 nucleotides encoding a polypeptide of 422 amino acid residues. The amino acid sequence has a high degree of homology with hamster (78%), mouse (76%), and rat (74%). XhoI and SacI sites restricted 1158 bp fragment of zokor ZP3 cDNA, excluding the signal sequence and transmembrane-like domain was cloned under the phage T7 promoterlac operator control in the pET-28a(+) vector. Recombinant pET-zokorZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strain BL21 (DE3). Optimum expression of r-ZP3 was observed at 28 degrees C, 1 mM IPTG and 2 h of inducing. The purified protein was tested by Western blot.

A counting method for density packed cells based on sliding band filter image enhancement.

Cell loss and addition is an important biological event in pathology, and it usually provides central information to the changes of biological activity in the histological sections. To develop a reliable and accurate cell counting tools in tissue section, in this paper, we proposed a novel cell nuclei detecting method based on the sliding band filter which is a member of convergence index family. We evaluated the accuracy and performance of our method on density packed retinal outer nuclear layer cell confocal multivariate fluorescence microscopy image datasets. The results show our proposed method exhibited an excellent performance with its accuracy compared with human manual counting. It is worth noting that the proposed cell counting method can clearly benefit for retinal detachment and reattachment visual diagnostics close related to cell loss and addition.

[Protective effect of Panax quinquefolium 20s-proto-panaxdiolsaponins on acute myocardial infarction in dogs].

OBJECTIVE: To study the protective effects of Panax quinquefolium 20s-protopanaxdiolsaponins extracted from leaves of P. quinquefolium (PQDS) on acute myocardial infarction(AMI) in dogs. METHOD: The parameters of myocardial infart size, the serum CK and LDH activity, myocardial metabolism, free radicals and coronary circulation etc were determined by using the model of ligation of LAD in the anaesthetized open-chest dogs. RESULT: In dogs treated with PQDS(in a dosage of 10 and 20 mg.kg-1 i.v. infusion), the myocardial infarct size, the activity of serum CK, LDH and the contents of serum FFA and LPO were decreased, whereas the activity of serum SOD and GSH-Px increased markedly. At the same time, myocardial blood flow was increased and coronary vascular resistance decreased significantly. CONCLUSION: PQDS has protective effect on myocardial ischemia by modifying metabolic dysfunction of FFA, inhibiting oxygen free radical mediated peroxidation of membrane lipids, enhancing endogenous antioxidase activity and increasing myocardial blood supply.

[Treatment of intracranial anterior circulatory aneurysms via keyhole approach].

OBJECTIVE: To evaluate the clinical effect of treatment of intracranial anterior circulatory aneurysm via the minimum invasive surgical technique keyhole approach. METHODS: Fifty three patients with intracranial anterior circulatory aneurysms were operated upon via keyhole approach from February to November 2000, 37 cases with ICA or MCA being operated upon via pterional approach, and 16 cases with AcoA or ACA aneurysms via frontal unilateral interhemispheric keyhole approach. A bone flap, 25 approximately 30 mm wide and 15 approximately 20 mm high, was created with a high-speed drill in craniotomy. The aneurysm was exposed through the lateral cerebral fissure or intercerebral fissure. RESULTS: The intracranial anterior circulatory aneurysms in 53 cases were successfully clipped via keyhole approach, of which 5 aneurysm bodies were resected and 12 cases underwent intraoperative accidental rupture. Postoperative angiography showed that all of the aneurysms were occluded. None of the cases died after operation. The morbidity of postoperative complication was 3.8%. No approach-related complication occurred. CONCLUSION: Keyhole approach helps to obtain the best operative effect for treatment of intracranial anterior circulatory aneurysm.

Interaction of insulin-like growth factor binding protein-4, Miz-1, leptin, lipocalin-type prostaglandin D synthase, and granulin precursor with the N-terminal half of type III hexokinase.

Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D synthase, and granulin precursor were identified as proteins interacting with the N-terminal half of mammalian Type III hexokinase (HKIII) in the yeast two-hybrid method. These interactions were confirmed by in vitro binding studies. All five of these proteins, and their mRNAs, were present in PC12 cells, as shown by immunoblotting and RT-PCR, respectively. All were coimmunoprecipitated from PC12 extracts with an antibody against HKIII, but not with anti-Type I hexokinase. Moreover, all of these proteins were coimmunoprecipitated using antileptin as precipitating antibody, indicating the existence of a macromolecular complex including these five proteins and HKIII. Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) reporter constructs gave a diffuse intracellular fluorescence. Cotransfection with leptin or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion protein, at intracellular sites coincident with localization of leptin-GFP or Miz-1-GFP reporter constructs.

The complete unique long sequence and the overall genomic organization of the GA strain of Marek's disease virus.

We have determined the DNA sequence of the unique long (UL) region and the repeat long (RL) region in the genome of serotype 1 GA strain of Marek's disease virus (MDV), a member of the alpha-herpesvirus family. With this information, the complete nucleotide sequence of GA-MDV is now known. The entire GA-MDV genome is predicted to be about 174 kbp in size, with an organization of TRL-UL-IRL-IRS-US-TRS, typical of a alpha-herpesvirus. The UL sequence contains 113,508 bp and has a base composition of 41.7% G + C. A total of 67 ORFs were identified completely within the UL region, among which 55 are homologous to genes encoded by herpes simplex virus-1. Twelve of them are unique with presently unknown functions. The sequence of RL reported here together with those published earlier reveal the major structural features of the RL. Virtually all of the ORFs encoded by RL are specific to serotype I of MDV. These ORFs are likely to contribute to some of the unique biological properties of MDV. Among the proteins encoded by MDV-specific ORFs are Meq, a jun/fos family of transcriptional factor implicated in transformation and latency, virus-encoded interleukin-8, a CXC chemokine, and pp38 and pp24, two phosphoproteins with undefined functions. There is also a putative lipase gene (LORF2) that has homologies in HPRS-24 (serotype II) strain of MDV and in various avian adenoviruses. An additional unique feature of MDV is the presence of long terminal repeat remnant sequences of avian retrovirus reticuloendotheliosis virus. These remnant sequences are derived from the U3-enhancer region through ancestral insertions by reticuloendotheliosis virus proviruses.

A novel NH(2)-terminal, nonhydrophobic motif targets a male germ cell-specific hexokinase to the endoplasmic reticulum and plasma membrane.

Although three germ cell-specific transcripts of type 1 hexokinase exist in murine male germ cells, only one form, HK1-sc, is found at the protein level. This single isoform localizes to three distinct structures in mouse spermatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum (Travis, A. J., Foster, J. A., Rosenbaum, N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998) Mol. Biol. Cell 9, 263-276). The mechanism by which one protein is targeted to multiple sites within this highly polarized cell poses important questions of protein targeting. Because the study of protein targeting in germ cells is hampered by the lack of established cell lines in culture, constructs containing different domains of the germ cell-specific hexokinase transcripts were linked to a green fluorescent protein and transfected into hexokinase-deficient M+R42 cells. Constructs containing a nonhydrophobic, germ cell-specific domain, present at the amino terminus of the HK1-SC protein, were targeted to the endoplasmic reticulum and the plasma membrane. Mutational analysis of this domain demonstrated that a complex motif, PKIRPPLTE (with essential residues italicized), represented a novel endoplasmic reticulum-targeting motif. Constructs based on another germ cell-specific hexokinase transcript, HK1-sa, demonstrated the specific proteolytic removal of an amino-terminal domain, resulting in a protein product identical to HK1-SC. Such processing might constitute a regulatory mechanism governing the spatial and/or temporal expression of the protein.

Identification of a novel PSD-95/Dlg/ZO-1 (PDZ)-like protein interacting with the C terminus of presenilin-1.

Presenilin-1 (PS-1) is the most causative Alzheimer gene product, and its function is not well understood. In an attempt to elucidate the function of PS-1, we screened a human brain cDNA library for PS-1-interacting proteins using the yeast two-hybrid system and isolated a novel protein containing a PSD-95/Dlg/ZO-1 (PDZ)-like domain. This novel PS-1-associated protein (PSAP) shares a significant similarity with a Caenorhabditis elegans protein of unknown function. Northern blot analysis revealed that PSAP is predominantly expressed in the brain. Deletion of the first four C-terminal amino acid residues of PS-1, which contain the PDZ domain-binding motif (Gln-Phe-Tyr-Ile), reduced the binding activity of PS-1 toward PSAP 4-fold. These data suggest that PS-1 may associate with a PDZ-like domain-containing protein in vivo and thus may participate in receptor or channel clustering and intracellular signaling events in the brain.

Identification and characterization of glycoprotein H of MDV-1 GA strain.

A 2439 bp open reading frame (ORF) was identified from the DNA sequence of BamHI-F and -K2 fragments of Marek's disease virus of serotype 1 (MDV-1) GA strain, which predicts an 813 amino acid polypeptide. This peptide is homologous to HSV-1 gH, and has typical glycoprotein features. There are nine potential N-linked glycosylation sites within the extracellular domain. A fragment of the gH ORF was cloned into pGEX vector in frame with glutathione S-transferase (GST) to produce a GST-gH fusion protein in Escherichia coli. The GST-gH fusion protein was used to develop gH monoclonal and polyclonal antibodies. Expression of gH was detected in duck embryo fibroblasts (DEFs) infected with MDV-1 GA strain by immunofluorescence assay (IFA) with these antibodies. Virus neutralization and plaque-forming inhibition analyses were conducted with the gH antiserum. There were no neutralization and plaque-forming inhibition activities of gH antiserum. Comparison of the DNA sequence of gH gene between GA and RB1B strains of MDV-1 revealed major difference in the upstream control elements of gH ORF.

Identification and structural analysis of a MDV gene encoding a protein kinase.

DNA sequence analysis of the BamHI-C fragment of Marek's Disease Virus (MDV) reveals the presence of a 513 amino acid open reading frame (ORF). This ORF codes for a protein with an estimated M(r) of 58,901. Comparison of the amino acid sequence with those available in the Swiss-Prot database indicates extensive homology with a protein kinase (PK) of herpes simplex virus (HSV) and varicella-zoster virus (VZV). In Northern blot hybridization, a transcript of 2.0 kb was detected in MDV (GA strain) infected duck embryo fibroblasts (DEFs). A portion of the ORF was expressed in Escherichia coli as a trpE-fusion protein and used to generate antiserum in New Zealand rabbits. This antiserum specifically detected a protein of 60 kDa in MDV serotype 1, 2 and 3 infected DEFs or chicken embryo fibroblasts (CEFs) by Western blot analysis. This ORF codes for a functional PK.

Combination of intraoperative embolization with surgical resection for treatment of giant cerebral arteriovenous malformation.

OBJECTIVE: To reduce the risk of surgical resection of giant arteriovenous malformation (AVM) (> 6.0 cm) and prevent normal perfusion pressure breakthrough (NPPB) for lowering the postoperative mortality. METHODS: During the operation under barbiturate anesthesia, the proximal end of the feeding arteries were ligated at first, and 0.5 ml isobutyl 12-cyanoacrylate (IBCA) with 0.5 ml 5% glucose was injected into the vessels towards the AVM, then the malformed vessels were resected totally. Postoperative digital subtraction angiography of the four vessels was performed in all patients. RESULTS: 50 patients with giant AVM survived after operation, only 6 (12.0%) had transient neurological dysfunction and 44 (88.0%) recovered after a follow-up of 6-36 months. No patient suffered from normal perfusion pressure breakthrough (NPPB). CONCLUSIONS: The embolization could block the arteriovenous shunts sufficiently to decrease the blood flow away from the normal areas of the brain so as to prevent the incidence of intra- and postoperative rebleeding, especially in NPPB. Therefore, the combination of intraoperative embolization with surgical resection is an effective strategy in the treatment of giant cerebral AVMs, which make it operable for those used to be regarded as inoperable cases.

GIS-based urban modelling: practices, problems, and prospects.

"This paper reviews the practices, problems, and prospects of GIS-based urban modelling. The author argues that current stand-alone and various loose/tight coupling approaches for GIS-based urban modelling are essentially technology-driven without adequate justification and verification for the urban models being implemented. The absolute view of space and time embodied in the current generation of GIS also imposes constraints on the type of new urban models that can be developed. By reframing the future research agenda from a geographical information science (GISci) perspective, the author contends that the integration of urban modelling with GIS must proceed with the development of new models for the informational cities, the incorporation of multi-dimensional concepts of space and time in GIS, and the further extension of the feature-based model to implement these new urban models and spatial-temporal concepts according to the emerging interoperable paradigm."

Structural determinants for the intracellular localization of the isozymes of mammalian hexokinase: intracellular localization of fusion constructs incorporating structural elements from the hexokinase isozymes and the green fluorescent protein.

Fusion constructs incorporating structural elements from mammalian isozymes of hexokinase, Types I-IV, in frame with sequence encoding the green fluorescent protein (GFP) have been made and expressed in hexokinase-deficient M + R 42 cells. Fusion proteins incorporating catalytically active regions from the Type II isozyme, or the entire Type IV sequence, were expressed in catalytically active form. The intracellular localization of the fusion proteins was determined using confocal microscopy. Fusion proteins including the N-terminal halves of the Type I or Type II isozymes were targeted to mitochondria, while the N-terminal half of the Type III isozyme did not confer mitochondrial targeting. The mitochondrial targeting signal was represented by the hydrophobic sequence at the extreme N-termini ("binding domain") of the Type I and Type II isozymes. Inclusion of the binding domain from the Type I isozyme was sufficient to confer mitochondrial binding on GFP itself as well as on constructs including the N-terminal half of Type III hexokinase. However, the Type I hexokinase binding domain was not sufficient to cause mitochondrial targeting of a construct containing the Type IV sequence. These results suggest that, although the binding domain is critical for mitochondrial targeting, other interactions involving an adjacent structure might also play a role. Fusion proteins including the N-terminal half of Type I hexokinase became dissociated from mitochondria under conditions favorable for accumulation of intracellular Glc-6-P. The 2-deoxy analog was much less effective than Glc in causing mitochondrial dissociation of the fusion construct, in accord with previous studies showing 2-deoxy-Glc-6-P to be much less effective than Glc-6-P at promoting release of Type I hexokinase from mitochondria. Dissociation, induced by formation of Glc-6-P or 2-deoxy-Glc-6-P, did not occur with the fusion protein including only the binding domain of Type I hexokinase. This is consistent with previous studies indicating that Glc-6-P-dependent dissociation results from binding of this ligand to a site in the N-terminal half of the enzyme, but which is not likely to be present in the small segment represented by the binding domain. These studies demonstrate the usefulness of this approach in defining structural elements involved in targeting hexokinase isozymes to specific subcellular locations and modulation of that intracellular location by perturbations of metabolic status.

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1.

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).

Affinity chromatography of trypsin using chitosan as ligand support.

Chitosan beads were prepared for use as affinity adsorbent carrier. The affinity ligand, chicken ovomucoid, was immobilized on the chitosan via a cross-linker, glutaraldehyde. The results showed that 60 mg chicken ovomucoid could be immobilized on l g chitosan, and the maximum binding capacity for trypsin was about 8.10(4) U/g dry adsorbent. The procedure for preparing the chitosan-based affinity adsorbents was much safer and simpler than when a Sepharose-based matrix was the support. Columns packed with the affinity adsorbents were employed for trypsin chromatography. The experimental results revealed that the affinity adsorbents possessed good mechanical strength and storage stability and could be also operated repeatedly. Chitosan was suitable for use as an affinity adsorbent support for laboratory-scale and large-scale purification.

Identification and characterization of a Marek's disease virus gene encoding DNA polymerase.

DNA sequence analysis revealed a gene encoding the Marek's disease virus (MDV) DNA polymerase (pol) within the BamHI-E fragment of the long unique region of the virus genome. Identification is based on an extensive amino acid homology between the MDV open reading frame and the DNA pol (UL30) of the herpes simplex virus. We describe here a 3540-base-pair fragment of the MDV DNA encoding 1180 amino acids with a M(r) of 133,920 daltons as the viral DNA pol gene, with the analysis of transcription and translation. In Northern blot hybridization, a transcript of 4.0 kb was detected in GA-MDV-infected duck embryo fibroblast (DEF) cells. An antiserum was generated in rabbit using TryE-pol fusion protein expressed in E. coli. This antiserum specifically immunoprecipitated a protein of 135 kD from lysates of MDV-GA-infected DEF cells. MDV DNA pol showed extensive homology to five distantly related herpesviruses: equine herpesvirus (EHV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV). Comparison of amino acid sequences among the herpesviruses highlights nine highly conserved regions. Three of the conserved regions are in the N-terminus in the 3'-5' exonuclease domains and the remaining six are in the C-terminus in the catalytic domains. The predicted structural characters are in good agreement with the published data on a number of human herpesvirus DNA pol. The identification of MDV DNA pol gene may lead to a better understanding of MDV replication.

[Effects of the leaves of Acanthopanax senticosus (Rupr. et Maxim.) Harms. on myocardial infarct size in acute ischemic dogs].

Effects of saponin isolated from the leaves of Acanthopanax senticosus (ASS) on myocardial infarct size were studied in acute ischemic dogs. The results showed that ASS (in a dosage of 25.50 mg/kg, iv) could significantly reduce the sizes of acute myocardial infarcts and decline the serum CK and LDH activity at 6h after ligation of LAD. It could also decrease the serum FFA levels at 3h and 6h after LAD occlusion.