Efficient production of fluorophore-labeled CC chemokines for biophysical studies using recombinant enterokinase and recombinant sortase.

Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti-inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti-chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion-tagged chemokine is produced in Escherichia coli, then efficient cleavage of the N-terminal fusion partner is carried out with lab-produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab-produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP-fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti-inflammatory therapeutic, showing a binding constant for vCCI:vMIP-fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP-fluor) can be used in competition assays with other chemokines and we report a Kd for vCCI:CCL17 of 14 µM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.

Comprehensive genomic analysis of primary malignant melanoma of the esophagus reveals similar genetic patterns compared with epithelium-associated melanomas.

Primary malignant melanoma of the esophagus (PMME) is an exceedingly rare disease with a poor prognosis. The etiology of PMME remains largely unknown and genetic characteristics are yet to be clarified, essential for identifying potential therapeutic targets and defining treatment guidelines. Here, we performed whole-exome sequencing on 47 formalin-fixed paraffin-embedded specimens from 18 patients with PMME, including 23 tumor samples, 6 metastatic lymph nodes, and 18 tumor-adjacent normal tissues. The genomic features of PMME were comprehensively characterized, and comparative genomic analysis was further performed between these specimens and 398 skin cutaneous melanomas (SKCM), 67 non-esophagus mucosal melanomas (NEMM), and 79 uveal melanomas (UVM). In the PMME cohort, recurrently mutated driver genes, such as MUC16, RANBP2, NRAS, TP53, PTPRT, NF1, MUC4, KMT2C, and BRAF, were identified. All RANBP2 mutations were putatively deleterious, and most affected samples had multipoint mutations. Furthermore, RANBP2 showed parallel evolution by multiregional analysis. Whole-genome doubling was an early truncal event that occurred before most driver mutations, except for in TP53. An ultraviolet radiation-related mutational signature, SBS38, was identified as specific to epithelial melanomas and could predict inferior survival outcomes in both PMME and SKCM patients. Comparing the mutational and copy number landscapes between PMME and other subtypes of melanoma revealed that PMME has a similar genomic pattern and biological characteristics to SKCM. In summary, we comprehensively defined the key genomic aberrations and mutational processes driving PMME and suggested for the first time that PMME may share similar genomic patterns with SKCM; therefore, patients with rare melanomas, such as PMME, may benefit from the current treatment used for common cutaneous melanoma.

Urchin-like magnetic microspheres for cancer therapy through synergistic effect of mechanical force, photothermal and photodynamic effects.

BACKGROUND: Magnetic materials mediated by mechanical forces to combat cancer cells are currently attracting attention. Firstly, the magnetic force penetrates deeper into tissues than the NIR laser alone to destroy tumours. Secondly, the synergistic effect of nano-magnetic-material characteristics results in a viable option for the targeted killing of cancer cells. Therefore, mechanical force (MF) produced by magnetic nanomaterials under low frequency dynamic magnetic field combined with laser technology is the most effective, safe and efficient tool for killing cancer cells and tumour growth. RESULTS: In this study, we synthesized novel urchin-like hollow magnetic microspheres (UHMMs) composed of superparamagnetic Fe3O4. We demonstrated the excellent performance of UHMMs for killing laryngocarcinoma cancer cells through mechanical force and photothermal effects under a vibrating magnetic field and near-infrared laser, respectively. The killing efficiency was further improved after loading the synthesised UHMMs with Chlorin e6 relative to unloaded UHMMs. Additionally, in animal experiments, laryngocarcinoma solid tumour growth was effectively inhibited by UHMMs@Ce6 through magneto-mechanic force, photothermal and photodynamic therapy. CONCLUSIONS: The biocompatibility and high efficiency of multimodal integrated therapy with the UHMMs prepared in this work provide new insights for developing novel nano therapy and drug loading platforms for tumour treatment. In vivo experiments further demonstrated that UHMMs/Ce6 are excellent tools for strongly inhibiting tumour growth through the above-mentioned characteristic effects.

A parametrically programmable delay line for microwave photons.

Delay lines that store quantum information are crucial for advancing quantum repeaters and hardware efficient quantum computers. Traditionally, they are realized as extended systems that support wave propagation but provide limited control over the propagating fields. Here, we introduce a parametrically addressed delay line for microwave photons that provides a high level of control over the stored pulses. By parametrically driving a three-wave mixing circuit element that is weakly hybridized with an ensemble of resonators, we engineer a spectral response that simulates that of a physical delay line, while providing fast control over the delay line's properties. We demonstrate this novel degree of control by choosing which photon echo to emit, translating pulses in time, and even swapping two pulses, all with pulse energies on the order of a single photon. We also measure the noise added from our parametric interactions and find it is much less than one photon.

Bronchial Basaloid Papillary Tumor of Uncertain Malignant Potential: A Case Report.

Bronchial papillomas are benign tumors, which can be divided into different subtypes based on the cellular features. So far, no bronchial papilloma with basaloid cell features has been reported. We report a bronchial basaloid papillary tumor in a 67-year-old woman. Tumor recurrence and malignant transformation were observed after a long-term follow-up. The clinical, histological, immunohistochemical, and genetic features were reappraised. The primary tumor was characterized by papillary growth pattern and basaloid neoplastic cells, only a small amount of neoplastic cells showed mature characteristics. The tumor originated from respiratory epithelium and had a low proliferation index by Ki-67. Keratin (KRT) 5/6 and KRT7 showed patchy or partial positivity. Myoepithelial markers were negative. P63 was diffusely positive, but it was negative in the small amount of tumor cells with mature characteristics. The common genetic alterations (EGFR, KRAS, BRAF V600E, HER2, and ALK) of lung cancers were not detected. However, tumor recurrence was observed in the mediastinum and esophagus 12 years after surgery. The recurrent tumor had a morphology overlapping with that of the primary tumor; however, it displayed significantly malignant characteristics. The recurrent tumor was not related to high-risk HPV. A high variant allele frequency was observed in tumor suppressor gene BRCA1, TP53, oncogene GNA11, and KIT, which were all missense mutations. Considering the bland features of the primary tumor and the fact of tumor recurrence and undisputed malignant transformation, the basaloid papillary tumor was considered a tumor with uncertain malignant potential.

Crowded environments tune the fold-switching in metamorphic proteins.

Metamorphic proteins such as circadian clock protein KaiB and human chemokine XCL1 play vital roles in regulating biological processes, including gene expression, circadian clock and innate immune responses, and perform distinct functions in living cell by switching different structures in response to cellular environment stimuli. However, it is unclear how complex and crowded intracellular environments affect conformational rearrangement of metamorphic proteins. Here, the kinetics and thermodynamics of two well-characterized metamorphic proteins, circadian clock protein KaiB and human chemokine XCL1, were quantified in physiologically relevant environments by using NMR spectroscopy, indicating that crowded agents shift equilibrium towards the inactive form (ground-state KaiB and Ltn10-like state XCL1) without disturbing the corresponding structures, and crowded agents have predominantly impact on the exchange rate of XCL1 that switches folds on timescales of seconds, but have slightly impact on the exchange rate of KaiB that switches folds on timescales of hours. Our data shed light on how metamorphic proteins can respond immediately to the changed crowded intracellular conditions that induced by environmental cues and then execute different functions in living cell, and it also enhances our understanding of how environments enrich the sequence-structure-function paradigm.

The Effect of Select SARS-CoV-2 N-Linked Glycan and Variant of Concern Spike Protein Mutations on C-Type Lectin-Receptor-Mediated Infection.

The SARS-CoV-2 virion has shown remarkable resilience, capable of mutating to escape immune detection and re-establishing infectious capabilities despite new vaccine rollouts. Therefore, there is a critical need to identify relatively immutable epitopes on the SARS-CoV-2 virion that are resistant to future mutations the virus may accumulate. While hACE2 has been identified as the receptor that mediates SARS-CoV-2 susceptibility, it is only modestly expressed in lung tissue. C-type lectin receptors like DC-SIGN can act as attachment sites to enhance SARS-CoV-2 infection of cells with moderate or low hACE2 expression. We developed an easy-to-implement assay system that allows for the testing of SARS-CoV-2 trans-infection. Using our assay, we assessed how SARS-CoV-2 Spike S1-domain glycans and spike proteins from different strains affected the ability of pseudotyped lentivirions to undergo DC-SIGN-mediated trans-infection. Through our experiments with seven glycan point mutants, two glycan cluster mutants and four strains of SARS-CoV-2 spike, we found that glycans N17 and N122 appear to have significant roles in maintaining COVID-19's infectious capabilities. We further found that the virus cannot retain infectivity upon the loss of multiple glycosylation sites, and that Omicron BA.2 pseudovirions may have an increased ability to bind to other non-lectin receptor proteins on the surface of cells. Taken together, our work opens the door to the development of new therapeutics that can target overlooked epitopes of the SARS-CoV-2 virion to prevent C-type lectin-receptor-mediated trans-infection in lung tissue.

The Antiviral Activity of the Lectin Griffithsin against SARS-CoV-2 Is Enhanced by the Presence of Structural Proteins.

Although COVID-19 transmission has been reduced by the advent of vaccinations and a variety of rapid monitoring techniques, the SARS-CoV-2 virus itself has shown a remarkable ability to mutate and persist. With this long track record of immune escape, researchers are still exploring prophylactic treatments to curtail future SARS-CoV-2 variants. Specifically, much focus has been placed on the antiviral lectin Griffithsin in preventing spike protein-mediated infection via the hACE2 receptor (direct infection). However, an oft-overlooked aspect of SARS-CoV-2 infection is viral capture by attachment receptors such as DC-SIGN, which is thought to facilitate the initial stages of COVID-19 infection in the lung tissue (called trans-infection). In addition, while immune escape is dictated by mutations in the spike protein, coronaviral virions also incorporate M, N, and E structural proteins within the particle. In this paper, we explored how several structural facets of both the SARS-CoV-2 virion and the antiviral lectin Griffithsin can affect and attenuate the infectivity of SARS-CoV-2 pseudovirus. We found that Griffithsin was a better inhibitor of hACE2-mediated direct infection when the coronaviral M protein is present compared to when it is absent (possibly providing an explanation regarding why Griffithsin shows better inhibition against authentic SARS-CoV-2 as opposed to pseudotyped viruses, which generally do not contain M) and that Griffithsin was not an effective inhibitor of DC-SIGN-mediated trans-infection. Furthermore, we found that DC-SIGN appeared to mediate trans-infection exclusively via binding to the SARS-CoV-2 spike protein, with no significant effect observed when other viral proteins (M, N, and/or E) were present. These results provide etiological data that may help to direct the development of novel antiviral treatments, either by leveraging Griffithsin binding to the M protein as a novel strategy to prevent SARS-CoV-2 infection or by narrowing efforts to inhibit trans-infection to focus on DC-SIGN binding to SARS-CoV-2 spike protein.

Sustained Delivery of the Antiviral Protein Griffithsin and Its Adhesion to a Biological Surface by a Silk Fibroin Scaffold.

The protein Griffithsin (Grft) is a lectin that tightly binds to high-mannose glycosylation sites on viral surfaces. This property allows Grft to potently inhibit many viruses, including HIV-1. The major route of HIV infection is through sexual activity, so an important tool for reducing the risk of infection would be a film that could be inserted vaginally or rectally to inhibit transmission of the virus. We have previously shown that silk fibroin can encapsulate, stabilize, and release various antiviral proteins, including Grft. However, for broad utility as a prevention method, it would be useful for an insertable film to adhere to the mucosal surface so that it remains for several days or weeks to provide longer-term protection from infection. We show here that silk fibroin can be formulated with adhesive properties using the nontoxic polymer hydroxypropyl methylcellulose (HPMC) and glycerol, and that the resulting silk scaffold can both adhere to biological surfaces and release Grft over the course of at least one week. This work advances the possible use of silk fibroin as an anti-viral insertable device to prevent infection by sexually transmitted viruses, including HIV-1.

Molecular genetic characteristics of thymic epithelial tumors with distinct histological subtypes.

BACKGROUND: Due to the low incidence and histological heterogeneity, the molecular features and underlying carcinogenic mechanisms of thymic epithelial tumors (TETs) are yet to be fully elucidated, especially for different subtypes of TETs. METHODS: Tumor tissue samples of 43 TETs with distinct histological subtypes were collected. We analyzed the molecular characteristics in different subtypes based on whole exome sequencing data. RESULTS: The mutational profiles of the different subtypes of TETs varied. Compared with thymomas, thymic carcinomas (TCs) had a higher mutation frequency of MYO16 (33% vs. 3%, p = 0.024) and a lower frequency of ZNF729 mutations (0% vs. 35%, p = 0.044). No significant difference was observed in the median tumor mutation burden across different subtypes. The value of copy number variation burden, weighted genome instability index, and the number of amplified segments were all higher in TCs than thymomas, and they also tended to be higher in B3 thymoma than in non-B3 thymomas, while they had no significant differences between B3 thymoma and TCs. Clustering analyses revealed that Wnt, MAPK, Hedgehog, AMPK, and cell junction assembly signaling pathways were exclusively enriched in non-B3 thymomas, lysine degradation pathway in B3 thymoma, and extracellular matrix-receptor (ECM-receptor) interaction, positive regulation of cell cycle process, and activation of innate immune response pathways in TCs. CONCLUSIONS: This study revealed distinct molecular landscapes of different subtypes of TETs, suggesting diverse pathogenesis of non-B3 thymomas, B3 thymomas, and TCs. Our findings warrant further validation in future large-scale studies and may provide a theoretical basis for potential personalized therapeutic strategies.

The binding and specificity of chemokine binding proteins, through the lens of experiment and computation.

Chemokines are small proteins that are critical for immune function, being primarily responsible for the activation and chemotaxis of leukocytes. As such, many viruses, as well as parasitic arthropods, have evolved systems to counteract chemokine function in order to maintain virulence, such as binding chemokines, mimicking chemokines, or producing analogs of transmembrane chemokine receptors that strongly bind their targets. The focus of this review is the large group of chemokine binding proteins (CBP) with an emphasis on those produced by mammalian viruses. Because many chemokines mediate inflammation, these CBP could possibly be used pharmaceutically as anti-inflammatory agents. In this review, we summarize the structural properties of a diverse set of CBP and describe in detail the chemokine binding properties of the poxvirus-encoded CBP called vCCI (viral CC Chemokine Inhibitor). Finally, we describe the current and emerging capabilities of combining computational simulation, structural analysis, and biochemical/biophysical experimentation to understand, and possibly re-engineer, protein-protein interactions.

Longitudinal genomic alternations and clonal dynamics analysis of primary malignant melanoma of the esophagus.

Primary malignant melanoma of the esophagus (PMME) is a rare gastrointestinal melanoma with a high rate of recurrence and metastasis. The standard of care for PMME has not been established yet due to a lack of understanding of its clinical and molecular pathogenesis. Thus, we performed genomic profiling on a recurrent PMME case to seek novel opportunities for the management of this rare disease. Between 2013 and 2016, 6 tissue samples including 3 from the primary tumors, 2 from the relapsed tumors, and 1 from a normal control were collected from a patient diagnosed with PMME and were subjected to whole-exome sequencing to track the dynamic genetic changes. Additionally, we also analyzed a cohort of 398 samples obtained from the TCGA skin cutanesous melanoma (TCGA-SKCM) dataset to assess the frequency and determine the clinical implications of genomic events found in the presented study. ARHGAP35 (p.L1022M) was the only mutation shared across temporal PMME lesions. The PMME samples showed higher levels of genetic instability and intra-tumor heterogeneity. They also shared several concordant copy number variations (CNV). All lesions were concordant with the evolution trajectory, and shrinkage of the founding clone caused the subclonal population to become dominant in PT1c, which was likely the reason behind metastatic seeding. ARHGAP35 mutations were found in 6% of the TCGA-SKCM cohort samples. The presence of the mutations was associated with poor progression-free survival (PFS) by both univariate and multivariate Cox regression analyses. Our study showed that the primary tumor clone disseminates earlier in PMME. This highlights the need to understand the mechanism involved in the early PMME recurrence to optimize treatment.

Design and evaluation of a multi-epitope assembly peptide (MEAP) against herpes simplex virus type 2 infection in BALB/c mice.

BACKGROUND: Human herpes simplex virus (HSV) 1 and 2 causes oral, ocular, or genital infections, which remains a significant health problem worldwide. HSV-1 and -2 infections in humans range from localized skin infections of the oral, ocular, and genital regions to severe and often disseminated infections in immunocompromised hosts. Epitope based vaccination is a promising mean to achieve protective immunity and to avoid infections with Human herpes simplex virus type 2 (HSV-2). METHODS: The twelve selected epitopes, six B cell epitopes from different glycoprotein of HSV-2 (amino acid residues 466-473 (EQDRKPRN) from envelope glycoprotein B, 216-223 (GRTDRPSA) from C, 6-18 (DPSLKMADPNRFR) from D, 483-491 (DPPERPDSP) from E, 572-579 (EPPDDDDS) from G and 286-295 (CRRRYRRPRG) from I glycoprotein of HSV-2), four CD4+ T cell epitopes (amino acid residues 21-28 (NLPVLDQL) from D, 162-177 (KDVTVSQVWFGHRYSQ) from B, 205-224 (KAYQQGVTVDSIGMLPRFIP) from D and 245-259 (KPPYTSTLLPPELSD) from D) and two CD8+ T cell epitopes (amino acid residues 10-20 (KMADPNRFRGK) from D and 268-276 (ALLEDPAGT) from D), are responsible for the elicitation of the neutralizing antibodies and cytotoxic T lymphocytes (CTLs) that impart protective immunity to the host. In this study, all above epitopes were inserted into the extracellular fragment (amino acid residues 1-290) of HSV-2 glycoprotein D to construct multi-epitope assembly peptides (MEAPs) by replacing some non-epitope amino acid sequences. The epitope independency of the MEAPs was predicted by three-dimensional software algorithms. The gene of the selected MEAP was expressed in E.coli BL21(DE3), and its protective efficacy against HSV-2 infection was assessed in BALB/c mice. RESULTS: The MEAP, with each inserted epitopes independently displayed on the molecule surface, was selected as candidate proteins. The results showed that the MEAP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. CONCLUSIONS: The MEAP provided complete protection against infection with HSV-2 in mice, which indicates that it might be a potential candidate vaccine against HSV-2.

Malignant Gastrointestinal Neuroectodermal Tumor in the Right Heart: A Report of an Extremely Rare Case Presenting With a Cardiac Mass.

Malignant gastrointestinal neuroectodermal tumor (GNET) is an extremely rare soft tissue sarcoma and has been designated as a new entity recently. At present, GNET virtually exclusively occurs in the gastrointestinal tract. Here we report a case of extra-GNET that arose in the right heart. A 62-year-old male complained of chest distress and breathing difficulty while lying down at night for over 1 month at admission. The radiological findings revealed an occupying lesion involving the right atrium and the right ventricle without any abdominal abnormalities. The patient then underwent a surgical resection. Microscopically, neoplastic cells proliferated in the pattern of nests and sheets with fibrous separation. Focal areas with cellular dyscohesion imparted a vague pseudopapillary pattern. These tumor cells were small to medium in size with fine chromatin and predominantly pale eosinophilic cytoplasm. The nuclei were typically round to oval with somewhat irregular contours and contained small nucleoli. The mitotic figures were easily found. Immunohistochemically, the neoplastic cells were positive for S100 and SOX-10 but negative for HMB-45, A103, and CD99. EWSR1-AFTF1 rearrangement was detected by fluorescence in situ hybridization and further confirmed by whole-transcriptome sequence analysis. The patient had pulmonary metastasis 8 months later and soon died of the disease. The overall survival of the patient was 20 months. In summary, we reported an extremely rare case of cardiac GNET, indicating that the location of GNET should not be confined to the GI tract as initially defined. Due to the lack of a specific effective treatment and the occurrence of early metastasis, cardiac GNET conferred a poor prognosis. More clinical and experimental studies are warranted to better manage this disease in the future.

Self-assembly of pH-sensitive random copolymers: poly(styrene-co-4-vinylpyridine).

A series of pH-sensitive random copolymers, poly(styrene-co-4-vinylpyridine) (PSVP), with molecular weight about 10,000 and 4-vinylpyridine molar contents of 19-53%, were conveniently synthesized by free radical polymerization. The copolymers experience the formation, swell, and dissociation of multichain nanoparticles when the pH of the aqueous solutions/dispersions changes from 5.1 to 1.0. The nanoparticles have hydrodynamic diameters around 100 nm, a spherical shape, and a relatively uniform structure in a pH range of about 5-3 and a multicore structure at lower pH. The random distribution of the building units causes some of the hydrophilic units, protonated 4-vinylpyridine groups, to be trapped inside the nanoparticles. So the hydrophobicity of the nanoparticles is tunable by changing the 4-vinylpyridine content in the copolymers. For the copolymers with higher 4-vinylpyridine molar content, the pH range in which the multichain nanoparticles form shifts to higher values, the multichain nanoparticles dissociate, and the copolymers form single-chain hydrophobic domains at low pH.

Companied P16 genetic and protein status together providing useful information on the clinical outcome of urinary bladder cancer.

SPEC P16/CEN3/7/17 Probe fluorescence-in-situ-hybridization (FISH) has become the most sensitive method in indentifying the urothelial tumors and loss of P16 has often been identified in low-grade urothelial lesions; however, little is known about the significations of other P16 genetic status (normal and amplification) in bladder cancer.We detected P16 gene status by FISH in 259 urine samples and divided these samples into 3 groups: 1, normal P16; 2, loss of P16; and 3, amplified P16. Meanwhile, p16 protein expression was measured by immunocytochemistry and we characterized the clinicopathologic features of cases with P16 gene status.Loss of P16 occurred in 26.2%, P16 amplification occurred in 41.3% and P16 gene normal occurred in 32.4% of all cases. P16 genetic status was significantly associated with tumor grade and primary tumor status (P = .008 and .017), but not with pathological tumor stage, overall survival, and p16 protein expression. However, P16 gene amplification accompanied protein high-expression has shorter overall survival compared with the overall patients (P = .023), and P16 gene loss accompanied loss of protein also had the tendency to predict bad prognosis (P = .067).Studies show that the genetic status of P16 has a close relation with the stages of bladder cancer. Loss of P16 is associated with low-grade urothelial malignancy while amplified P16 donotes high-grade. Neither P16 gene status nor p16 protein expression alone is an independent predictor of urothelial bladder carcinoma, but combine gene and protein status together providing useful information on the clinical outcome of these patients.

Differences in Clinicopathology of Early Gastric Carcinoma between Proximal and Distal Location in 438 Chinese Patients.

Early gastric carcinoma (EGC) in Chinese patients remains poorly understood and endoscopic therapy has not been well established. Here, we compared endoscopic and clinicopathologic features between early proximal gastric carcinoma (PGC, n = 131) and distal gastric carcinoma (DGC, n = 307) in consecutive 438 EGCs diagnosed with the WHO criteria. By endoscopy, PGCs showed protruding and elevated patterns in 61.9%, while depressed and excavated patterns in 33.6%, which were significantly different from those (32.6% and 64.5%) in DGCs. PGCs were significantly smaller (1.9 cm in average, versus 2.2 cm in DGCs), invaded deeper (22.9% into SM2, versus 13% in DGCs), but had fewer (2.9%, versus 16.7% in DGCs) lymph node metastases. Papillary adenocarcinoma was significantly more frequent (32.1%, versus 12.1% in DGCs), as were mucinous and neuroendocrine carcinomas, carcinoma with lymphoid stroma (6.9%, versus 1.6% in DGCs); but poorly cohesive carcinoma was significantly less frequent (5.3%, versus 35.8% in DGCs). The overall 5-year survival rate was 92.9% in EGCs, and PGC patients showed shorter (42.4 months, versus 48.3 in DGCs) survival. Papillary and micropapillary adenocarcinomas and nodal metastasis were independent risk factors for worse survival in EGCs. EGCs in Chinese were heterogeneous with significant differences in endoscopy and clinicopathology between PGC and DGC.

Clinicopathological characterisation of small (2 cm or less) proximal and distal gastric carcinomas in a Chinese population.

Clinicopathological characteristics of small gastric carcinoma have not been well defined in Chinese patients. The aim of this study was to investigate and compare small proximal (PGC, n = 111) with distal (DGC, n = 202) gastric carcinoma in 313 consecutive surgically resected small (≤2 cm) gastric carcinomas diagnosed with the WHO criteria. PGC patients were significantly older (average age 63 years versus 59 in DGCs) with a male/female ratio of 3:1. Most tumours were clustered along the lesser curvature (74% in PGCs and 65% in DGCs). Compared to DGCs, PGCs showed a protruded gross pattern significantly more frequently and were significantly better differentiated with a significantly wider histomorphological spectrum. Surprisingly, PGCs were composed of significantly fewer signet-ring cell carcinomas (1% versus 16% in DGCs) but were significantly more deeply invasive, compared to DGCs. Lymph node metastasis was detected in 23% overall, but was significantly less frequent in PGCs (16%) than in DGCs (26%) (p < 0.05). However, the difference in survival between the two groups was not statistically significant. Our results demonstrate that in Chinese patients, PGCs display distinct clinicopathological characteristics, compared to DGCs.

[Eucaryotic expression and bioactivity analysis of the recombinant HSV-gD1].

Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5 x10(3). The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.