Simple technique for high-throughput marking of distinguishable micro-areas for microscopy.

Today's (nano)-functional materials, usually exhibiting complex physical properties require local investigation with different microscopy techniques covering different physical aspects such as dipolar and magnetic structure. However, often these must be employed on the very same sample position to be able to truly correlate those different information and corresponding properties. This can be very challenging if not impossible especially when samples lack prominent features for orientation. Here, we present a simple but effective method to mark hundreds of approximately 15×15 µm sample areas at one time by using a commercial transmission electron microscopy grid as shadow mask in combination with thin-film deposition. Areas can be easily distinguished when using a reference or finder grid structure as shadow mask. We show that the method is suitable to combine many techniques such as light microscopy, scanning probe microscopy and scanning electron microscopy. Furthermore, we find that best results are achieved when depositing aluminium on a flat sample surface using electron-beam evaporation which ensures good line-of-sight deposition. This inexpensive high-throughput method has several advantageous over other marking techniques such as focused ion-beam processing especially when batch processing or marking of many areas is required. Nevertheless, the technique could be particularly valuable, when used in junction with, for example focused ion-beam sectioning to obtain a thin lamellar of a particular pre-selected area.

On the identity of Oberea acuta Gressitt, 1951, O. bisbipunctata Pic, 1916, O. notata Pic, 1936 and O. shimomurainbsp;Kurihara amp; N. Ohbayashi, 2007 br>(Coleoptera, Cerambycidae, Lamiinae) with a new synonym.

Oberea acuta Gressitt, 1951 is redescribed, the diagnosis of O. notata Pic, 1936 and O. bisbipunctata Pic, 1916 were provided based on examination of type material and additional specimens. Illustrations of habitus and genitalia are provided, and a key to separate these three species is presented. Their distribution is critically examined, notably that of O. bisbipunctata, whose previous records in China are invalidated. Oberea shimomurai Kurihara N. Ohbayashi, 2007 is considered a junior synonym of Oberea notata Pic, 1936.

Megarthrus of China. Part 3. Comments on the occurrence of Megarthrus nitidulus Kraatz, 1857 in China, and description of a new species, with notes on teratology and phoretic acari in the genus (Coleoptera: Staphylinidae: Proteininae).

A new species, Megarthrus heise Zhang, Cuccodoro, Chen Liu sp. nov., resembling Megarthrus nitidulus Kraatz, 1857 is described from Qinghai, Sichuan, Gansu, Shaanxi, Shanxi provinces and Beijing in China. The record of M. nitidulus in China was apparently based on a misidentification, and the species is thus removed from the list of Chinese Megarthrus. Phoresy of acari and teratological modification of the abdomen are also reported for the first time in the genus.

Primary types of longhorned beetles (Coleoptera, Cerambycidae, Vesperidae and Disteniidae) of Southwest University (SWU).

The primary types of longhorned beetles of Southwest University (SWU) are catalogued and figured. The original combination, current name, type locality, date and collector of type specimen are verified and presented. There are 69 holotypes and 86 paratypes in SWU. These 155 type specimens belong to three families, 51 genera, 71 species, of which 8 are in Disteniidae, 3 in Vesperidae, 3 in Prioninae, 12 in Lepturinae, 15 in Cerambycinae, and 30 in Lamiinae. A new synonym is proposed: Paraleprodera insidiosa (Gahan, 1888) = Paraleprodera bimaculata Wang Chiang, 2000 syn. nov.

Total Flavonoids Isolated from Diospyros kaki L. f. Leaves Induced Apoptosis and Oxidative Stress in Human Cancer Cells.

BACKGROUND/AIM: Persimmon (Diospyros kaki L.) leaves are popular as a tea infusion in Asia and their main active ingredients are flavonoids. The present study aimed to explore the anticancer properties of flavonoids isolated from persimmon leaves (PLF). MATERIALS AND METHODS: We investigated the in vitro anti-proliferative activity of PLF against several human cancer cell lines. Apoptosis and intracellular reactive oxygen species (ROS) induced by PLF were accessed using high-content analysis with florescent staining. The ability of PLF to scavenge free radicals was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. RESULTS: PLF demonstrated significant inhibition of proliferation of liver, breast, and colorectal cancer cells in vitro. PLF induced apoptosis and increased intracellular ROS levels in HCT116 (colorectal cancer) and HepG2 (liver cancer) cells. In addition, PLF showed strong free radical scavenging ability. CONCLUSION: The anti-proliferation activity of PLF against cancer cells was related to the induction of apoptosis and oxidative stress.

Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase.

The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

Two new species of the genus Trachylophus Gahan, 1888 (Coleoptera, Cerambycinae, Cerambycini) from China.

Two new species of the genus Trachylophus Gahan, 1888, T. tianmuensis sp. nov. and T. chiangi sp. nov. from China are described. The adult habitus and male genitalia of the two new species are described in detail.

Coloration variability of the Oriental genus Agnorimus Miyake amp; Iwase, 1991 (Coleoptera: Scarabaeidae, Cetoniinae) with synonymic notes.

Based on an examination of a large number of specimens, considerable variability of the elytral coloration patterns and the parameres of the Asian genus Agnorimus Miyake Iwase, 1991 are observed; and the taxonomic status of all four described taxa have been re-considered. The result shows that only A. pictus (Moser, 1902) can be recognized as valid species, while A. hayashii Miyake Iwase, 1991 new synonym is proposed as a new junior synonym of it. The geographic distribution of this genus is significantly expanded, and most of the distributional records are illustrated in a map with intraspecific variations indicated.

Simultaneous acute nephrosis and hepatitis in secondary syphilis.

Simultaneous occurrence of acute nephrosis and hepatitis in secondary syphilis is rare. We report a 24-year-old man who presented with sudden onset of nephrotic syndrome, acute hepatitis, and skin lesions associated with secondary syphilis. A renal biopsy demonstrated electron-dense deposits located in the subepithelial, mesangial and intramembranous areas under electron microscopy. Light microscopy revealed a mild increase in mesangial matrix with normal glomerular cellularity. Immunofluorescent examination showed granular deposition of IgG and IgA along the glomerular capillary basement membrane without deposits of complements. These morphological changes differed from those reported previously. All, the heavy proteinuria, disturbances of liver function, and skin lesions resolved after 4-week treatment.

The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

Taxonomic review of the Oriental flower beetle Coilodera penicillata species complex (Coleoptera: Scarabaeidae: Cetoniinae).

The taxonomy of the Coilodera penicillata Hope, 1831 (Coleoptera: Scarabaeidae: Cetoniinae) species complex, which is recognized by the black body and elytra with larger tomentose maculae, is revised. Six Indochinese species assigned to this complex, including C. grandimaculata new species from Tibet, China. Coilodera nigroscutellaris Moser, 1902 revised status and C. formosana Moser, 1910 revised status formerly treated as two subspecies of C. penicillata Hope, 1831 are elevated to species rank. The distribution of C. penicillata in China is confirmed based on examination of specimens from Yunnan and Tibet, and almost all previous Chinese literature records should be referred to C. nigroscutellaris. Coilodera dives (Gory & Percheron, 1833) revised status previously synonymized with C. penicillata is herein considered as a valid species based on the examination of types and additional specimens, and C. miksici Antoine, 1986 new synonymy is regarded as a junior synonym of C. dives. A lectotype is designated for Coilodera dives (Gory & Percheron, 1833). The record of C. mearesii (Westwood, 1842) in southeastern China is attributed to a misidentification of C. nigroscutellaris, and it is here newly recoreded from Tibet near the Nepal-China border. Diagnosis characters and intraspecific variations are illustrated for all six species, new distribution records and a key to species are also presented. Ecological information is provided for all species.

Clinical treatment of gastrinoma: A case report and review of the literature.

Gastrinoma is a gastrin-secreting tumor that is associated with Zollinger-Ellison syndrome. The majority of cases occur in the pancreas, followed by the duodenum. Early diagnosis is difficult due to the relative rarity of the tumor and the lack of specific symptoms. In the current study, a 68-year-old female patient presented at the Second Affiliated Hospital, Zhejiang University (Hangzhou, China) due to intermittent abdominal pain and watery diarrhea. The patient was treated by surgical resection and was pathologically diagnosed with a well-differentiated pancreatic neuroendocrine tumor (gastrinoma; grade 1). No evidence of recurrence was observed during 1 year of follow-up. Furthermore, a review of the Chinese literature was performed, which analyzed an additional 17 published cases of gastrinoma. The tumor size ranged between 0.5×0.5 cm and 7.5×6.3×5.1 cm. The pancreas was the most common site of occurrence, accounting for 72% (13/18) of cases, followed by the duodenum (28%; 5/18). The most common initial symptom was abdominal pain (89%; 16/18), followed by diarrhea (56%; 10/18). In 18 cases, including the present case and 17 previous cases, the level of gastrin ranged between 137 and 1,550 pg/ml (normal range, 5-100 pg/ml). Of the 17 previous cases, 11 patients underwent surgery and 6 patients received conservative therapy due to metastasis or patient choice. Overall, gastrinoma remains a rare disease. Complete removal of the lesion is the standard curative treatment and conservative treatment is only recommended for patients unsuitable for surgery or for those with widespread metastasis.

A unique cathepsin-like protease isolated from CV-1 cells is involved in rapid degradation of retinoblastoma susceptibility gene product, RB, and transcription factor SP1.

The regulation of transcription factors by kinase or phosphatase has been well-described. However, little is known about the inactivation of transcription factors or the nuclear regulators by proteolytic degradation. In this report, we purified a specific protease, SPase, from nuclear extracts of the green monkey kidney cell line, CV-1. Studies of biochemical characteristics and substrate specificity indicated that SPase is a cathepsin B-like cysteinyl protease. However, the two tryptic peptide sequences derived from the purified SPase are either identical or highly homologous to those of human cathepsin L, and furthermore, SPase shares immunoreactivity with both anti-human cathepsin L and anti-mouse cathepsin L antibody. The SPase was shown to be localized in both cytoplasm and nucleus when subcellular compartments of CV-1 cells were fractionated. Transcription factor, SP1, and retinoblastoma susceptible gene product, RB, are substrates of SPase while other nuclear factors such as c-Jun and c-Fos are not. These results implied that SPase plays an integral role in regulating a set of proteins in the nuclei. In vivo treatment of CV-1 cells with cysteinyl protease inhibitor, E-64d, protected RB from degradation. SPase failed to degrade underphosphorylated RB present in TPA induced terminally differentiated HL-60 or U937 cells. Phosphorylation of RB may cause conformational changes, thus facilitating proteolytic digestion. These observations suggest that an alternative pathway inactivates the function of RB in controlling cell growth. Therefore, a possible role of SPase may be to affect the stability of important regulators involved in controlling cellular proliferation and differentiation.

Coxsackievirus A16 infection stimulates imbalances of T cells in children.

Immune reaction plays a crucial role in the regulation of the progression of Coxsackievirus A16 (CA16)-infected hand, foot and mouth disease (HFMD). However, no details of T-cell subset frequency or imbalance during the CA16 infection process have been revealed. In the present study, whether CA16-induced HFMD changes the frequency of different T-cell subsets and associated immune mediators was determined in children. The results indicate that the percentages of Th1 and Tc1 cells were significantly increased in children with HFMD compared with those in healthy children. In addition, the Th1/Th2 ratio and interferon (IFN)-γ levels were significant higher in children with HFMD. Furthermore, the percentage of Th17 cells and the Th17/Treg ratio as well as interleukin (IL)-17A levels were higher in HFMD cases. In conclusion, the present study demonstrated the dysregulation of T-cell subsets following CA16 infection. The Th1/Th2 and Th17/Treg ratios were imbalanced following infection. Also, the imbalance Th1/Th2 and Th17/Treg ratios contributed to the increased levels of IFN-γ and IL-17A. Based on this information, the present study provides new insights for the future study of CA16-induced HFMD and offers new data of diagnostic and therapeutic value for CA16 infection.

Ulinastatin enhances the therapeutic effect of intraperitoneal lavage on severe acute pancreatitis in rats.

The present study investigated the therapeutic effect of peritoneal lavage with ulinastatin on the outcome of rats with severe acute pancreatitis (SAP). A total of 110 male Wistar rats were randomly divided into the following groups: Control (C), SAP model (SAP), saline lavage (SL), intravenous ulinastatin (IU) and low-dose (LUL), medium-dose (MUL), high-dose (HUL) and ultrahigh-dose (UHUL) ulinastatin lavage. The treatments were performed immediately subsequent to the establishment of the SAP model. Intraperitoneal lavage with or without ulinastatin was performed for 3 h. Survival time was recorded for 12 h and the median survival time was calculated. Histopathological analyses, and measurements of plasma amylase and lipase levels were performed. Blood pH, lactic acid and base excess were also detected. The LUL, MUL, HUL, UHUL and IU groups showed an increase in the median survival time compared with the SAP group, with the maximal effect observed in the MUL group (P<0.01). The SL, MUL and IU groups showed a reduced activity of amylase and lipase compared with the SAP group. The SL (P<0.01) and the MUL groups (P<0.01) additionally showed a reduction in the lactic acid in arterial blood relative to the SAP group but the IU group did not. The MUL group showed greater improvements in pH (P<0.01) and base excess (P<0.01) versus the SAP group than the SL and IU groups. Furthermore the MUL group demonstrated a more marked reduction in the histological changes in necrosis, edema and inflammation compared with the SL and IU groups. Intraperitoneal lavage with ulinastatin significantly improves the prognosis of SAP in rats.

Malignant conversion of a solitary squamous cell papilloma in the trachea treated by radiotherapy: A case report.

The present study described a rare case of malignant conversion of a solitary papilloma in the trachea, in addition to reviewing the current literature. A 54-year-old male presented with a cough, sputum and dyspnea. Chest computed tomography revealed a mediastinal soft-tissue mass in the trachea. A tumor biopsy, performed using a flexible bronchofiberscope, identified a squamous cell papilloma with moderate to severe atypical hyperplasia, as well as the formation of squamous cell carcinoma. Polymerase chain reaction analysis did not detect presence of human papilloma virus in the tumor. The patient was subsequently treated with radiotherapy and no evidence of recurrence was observed during a two-year follow-up period. To the best of our knowledge, this is the first study to describe the malignant conversion of a solitary papilloma in the trachea, which was subsequently treated with radiotherapy.

Amplification and protein expression of androgen receptor gene in prostate cancer cells: Fluorescence in situ hybridization analysis.

Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and the second highest cause of cancer-related mortality in adult males worldwide. PCa is highly dependent upon androgen receptor (AR) signaling for cell proliferation and survival. The AR therefore plays a vital role in the development and function of normal and malignant prostate cells or PCa recurrence. The present study aimed to examine the ubiquity of AR amplification in PCa recurrence, even in the absence of androgen. For this purpose, specimens were collected from 37 patients. The amplification of AR and the number of X chromosomes were determined by two-colored fluorescence in situ hybridization analysis. The automated image analysis was used to determine the protein expression of AR. Clinical characteristics and survival in patients whose tumors showed or did not show AR amplification and in X-chromosome polysomy with PCa recurrence has also been compared. The results showed that >35% of patients (13 specimens) exhibited AR amplification. It was also observed that AR was immunostained more intensely in the tumors with amplified AR compared with those tumors with non-amplified AR. This study demonstrated an influential role of AR in tumor growth and progression even after the deprivation of androgen, as well as showing the potential contribution of AR amplification to AR activation even in the relative absence of androgen.

Management of retroperitoneal liposarcoma: A case report and review of the literature.

Retroperitoneal liposarcoma is a rare tumor with an incidence of 2.5 per million individuals. Early diagnosis is difficult as there is an absence of specific clinical presentations. The present case study reports a patient diagnosed with retroperitoneal liposarcoma who was treated by complete surgical resection and relapsed 3 months following the surgery. In addition, the clinical data of 14 patients with retroperitoneal liposarcoma were reviewed and analyzed. The mean age of the 14 patients at presentation was 54.1 (range, 36-73 years) and 5/14 patients experienced recurrence, ranging between 1 and 10 times. Of the 12 cases that reported histological subtypes, 7 were well-differentiated liposarcoma, 2 were dedifferentiated liposarcoma, 2 were myxoid liposarcoma and 1 was mixed subtype. All the patients underwent complete resection and 5 received combined multiple organs resection (3 nephrectomy, 1 sigmoid colon and 1 multiple visceral organs). However, no patients received chemotherapy or radiotherapy. In conclusion, retroperitoneal liposarcoma is a rare disease with a high rate of recurrence. Complete resection is the predominant treatment and combined resection of adjacent organs is occasionally necessary.

Effect of peritoneal lavage with ulinastatin on the expression of NF-κB and TNF-α in multiple organs of rats with severe acute pancreatitis.

The aim of the present study was to investigate the effect of peritoneal lavage with ulinastatin on the expression levels of nuclear factor κB (NF-κB) and tumor necrosis factor (TNF)-α in multiple organs of rats with severe acute pancreatitis (SAP). Male Wistar rats were randomly divided into the following groups: Sham-operated (C), SAP model (SAP), saline lavage (SL), intravenous ulinastatin (IU) and peritoneal lavage with ulinastatin (UL). The SAP model was induced by the retrograde infusion of 5% sodium taurocholate into the biliopancreatic ducts of the rats. Intraperitoneal lavage or injection was performed immediately following the establishment of the SAP model in groups SL, IU and UL. Intraperitoneal lavage with or without ulinastatin was performed for 3 h. The survival time of half of the rats in each group was recorded over a 12-h period. At 3 h after the induction of SAP, inflammatory mediators and the expression levels of NF-κB and TNF-α in multiple organs of the rats in each group were also detected. The survival rates of the rats in group UL at 6 h and 9 h were increased compared with those in group SAP, and were also higher than that in groups SL and IU. The levels of serum inflammatory mediators were effectively reduced in groups SL, IU and UL, the greatest effects were observed in group UL. The expression levels of NF-κB and TNF-α in multiple organs were significantly lower in group UL compared with other groups. Intraperitoneal lavage with ulinastatin significantly ameliorated the inflammatory reaction and inhibited NF-κB and TNF-α expression in multiple organs of SAP model rats.

Early peritoneal lavage with ulinastatin improves outcome and enhances multi-organ protection in a model of severe acute pancreatitis.

The aim of this study was to investigate the effect of early peritoneal lavage with ulinastatin on the outcome of a rat model of severe acute pancreatitis (SAP). A total of 80 male Wistar rats were randomly divided into the following groups: Sham-operated (C), SAP model (M), saline lavage (SL), intravenous ulinastatin (IU), early ulinastatin lavage (EUL) and late ulinastatin lavage (LUL). Intraperitoneal lavage or injection were performed immediately subsequent to the establishment of the SAP model in groups SL, IU and EUL and 3 h later in group LUL. Intraperitoneal lavage with or without ulinastatin was performed for 3 h. The survival time of the rats in groups C, M, EUL and LUL was recorded over a 12-h period and the median survival time was calculated. At 3 h after the induction of SAP, histopathological analyses were performed and the biochemical parameters of groups C, M, SL, IU and EUL were assessed. Groups EUL and LUL exhibited an increased median survival time compared with Group M, with the survival time of the rats in group EUL markedly longer than that in the group LUL rats. Group SL, IU and EUL rats were found to have reduced plasma activities of amylase, lipase, aspartate transaminase and alanine transaminase, with the biggest change observed in the group EUL rats. Furthermore, the intervention in groups SL and EUL was more effective at reducing creatinine and urea levels than that in group IU. Rats in group EUL exhibited a greater inhibition of the SAP-induced increase in troponin T levels than rats in groups SL and IU. The pathological severity scores of the pancreas, liver, kidney and lung in group EUL were significantly lower than those in groups M and better than those in groups SL and IU. In conclusion, early intraperitoneal lavage with ulinastatin significantly improves the median survival time and protects multi-organ function in an SAP model.