[Research progress of common respiratory virus receptor binding sites].

Respiratory viral infections are an important public health problem worldwide, with complex mechanisms of infection, and the key to infection lies in the specific binding between respiratory viruses and receptors. This article provides an overview of the progress in the study of receptors for respiratory viruses, such as coronavirus and influenza virus (IV), with a focus on the binding sites of receptors such as angiotensin-converting enzyme 2 (ACE2) and sialic acid (SA) to respiratory viruses and the role of receptor diversity in respiratory viral infections. An in-depth study of the binding sites between viruses and receptors will help to understand the molecular mechanism of respiratory viral infections and provide a theoretical basis for disease prevention and control and the development of new therapeutic targets.

[Minocycline inhibits formalin-induced inflammatory pain and the underlying mechanism].

OBJECTIVE: To unravel the underlying mechanism of minocycline in formalin-induced inflammatory pain, and to investigate the effects of minocycline on synaptic transmission in substantia gela-tinosa (SG) neurons of rat spinal dorsal horn. METHODS: Behavioral and immunohistochemistry experiments: 30 male Sprague-Dawley (SD) rats (3-5 weeks old) were randomly assigned to control (n=8 rats), model (n=8 rats), saline treatment model (n=6 rats) and minocycline treatment model (n=8 rats) groups. The control group was subcutaneously injected with normal saline on the right hindpaws. Acute inflammatory pain model was established by injecting 5% (volume fraction) formalin into the right hindpaws. The rats in the latter two groups received intraperitoneal injection of saline and minocycline 1 h before the formalin injection, respectively. The time of licking and lifting was recorded every 5 min within 1 h after the subcutaneous injection of normal saline or formalin for all the groups, which was continuously recorded for 1 h. One hour after the pain behavioral recording, the spinal cord tissue was removed following transcardial perfusion of 4% paraformaldehyde. The expression of c-Fos protein in spinal dorsal horn was observed by immunohistochemistry. Electrophysiological experiment: In vitro whole-cell patch-clamp recordings were performed in spinal cord parasagittal slices obtained from 26 male SD rats (3-5 weeks old). Two to five neurons were randomly selected from each rat for patch-clamp recording. the effects of minocycline, fluorocitrate and doxycycline on spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) of SG neurons were investigated. RESULTS: Compared with the control group, both the licking and lifting time and the expression of c-Fos protein in ipsilateral spinal dorsal horn of the model group were significantly increased. Intraperitoneal injection of minocycline largely attenuated the second phase of formalin-induced pain responses (t=2.957, P<0.05). Moreover, c-Fos protein expression was also dramatically reduced in both the superficial lamina (I-II) and deep lamina (III-IV) of spinal dorsal horn (tI-II=3.912, tIII-IV=2.630, P<0.05). On the other side, bath application of minocycline significantly increased the sIPSCs frequency to 220%±10% (P<0.05) of the control but did not affect the frequency (100%±1%, t=0.112, P=0.951) and amplitude (98%±1%, t=0.273, P=0.167) of sEPSCs and the amplitude (105%±3%, t=0.568, P=0.058) of sIPSCs. However, fluorocitrate and doxycycline had no effect on the frequency [(99%±1%, t=0.366, P=0.099); (102%±1%, t=0.184, P=0.146), respectively] and amplitude [(98%±1%, t=0.208, P=0.253); (99%±1%, t=0.129, P=0.552), respectively] of sIPSCs. CONCLUSION: Minocycline can inhibit formalin-induced inflammatory pain and the expression of c-Fos protein in spinal dorsal horn. These effects are probably due to its enhancement in inhibitory synaptic transmission of SG neurons but not its effect on microglial activation or antibiotic action.

[Spinal analgesic mechanism of minocycline in formalin-induced inflammatory pain].

Objective: To investigate the spinal analgesic mechanism of minocycline in formalin-induced inflammatory pain. Methods: Behavioral test: Male Sprague-Dawley rats(3-5-week old) were randomly assigned into four groups: control, model, vehicle-controlled and minocycline group. Ten percent neutral formalin was injected subcutaneously into the right hind paw dorsum of the rats in model, vehicle-controlled and minocycline group. Normal saline was injected subcutaneously into the right hind paw dorsum of the rats in control group. Before 1 h of formalin injection, the rats in vehicle-controlled and minocycline group received intraperitoneal injection of saline and minocycline, respectively. Licking and lifting time was observed as the behavior results of inflammatory pain. Electrophysiologic experiment: In vitro spinal cord parasagittal slices were prepared from the same rats as above. The effect of minocycline on spontaneous inhibitory postsynaptic currents(sIPSCs) of substantia gelatinosa(SG) neurons was observed using whole-cell patch-clamp technique. Results: Compared with the control group, the licking and lifting time in the model group was significantly increased. Compared with the vehicle-controlled group, the licking and lifting time in the minocycline group was significantly decreased. Minocycline significantly increased the frequency(t=9.32, P<0.05)but not the amplitude(t=1.54, P>0.05) of sIPSCs of SG neurons, the frequency of sIPSCs of control and minocycline group were (2.5±0.3)Hz and (5.2±0.6)Hz, respectively. When calcium was removed from the extracellular solution, the frequency before and after minocycline perfusion were (0.9±0.1)Hz and (0.9±0.1)Hz, respectively, the amplitude before and after minocycline perfusion were (18.2±0.7)pA and (18.5± 0.6)pA, respectively, the difference of frequency or amplitude was not statistically significant(t=0.32, 0.82, all P>0.05). However, minocycline still increased the frequency of sIPSCs when glutamate receptor antagonists 6-Cyano-7-nitroquinoxaline-2, 3-dione(CNQX) and D-(-)-2-Amino-5-phosphonopentanoic acid(APV) were included in extracellular solution(t=13.51, P<0.05), the frequency of sIPSCs were (2.0±0.1)Hz and (4.3±0.4)Hz, respectively. Minocycline still increased the frequency of IPSCs when voltage-gated sodium channel blocker tetrodotoxin(TTX) were included in extracellular solution(t=8.67, P<0.05), the frequency of IPSCs were (2.2±0.2)Hz and (5.2±0.5)Hz. Conclusion: Minocycline can attenuate formalin-induced inflammatory pain which may be associated with its increase in the inhibitory synaptic transmission of SG neurons.

Cloning, characterization and expression analysis of coagulation factor II gene in grass carp (Ctenopharyngodon idella).

Here, we characterized the structure and function of the coagulation factor II (FII) gene in grass carp and determined its role in coagulation mechanisms. The FII gene EST was obtained using a constructed splenic transcriptome database; the full-length FII gene sequence was obtained by 3' and 5' RACE. The open reading frame (ORF) of FII was cloned and the full-length gene was found to be 1718 bp, with an ORF of 1572 bp; the gene contained a 25 bp 5'-untranslated region (UTR) and 108 bp 3'-UTR. The ORF encoded 524 amino acids, including 74 alkaline amino acids (arginine and lysine) and 69 acidic amino acids (aspartic acid and glutamic acid). The theoretical pI was 6.22. The calculated instability index (II) was 39.81, indicating that FII was a stable protein; the half-life period was predicted to be approximately 30 h. Amino acid sequence comparisons indicated that grass carp FII showed most similarity (71%) to FII of Takifugu rubripes, followed by Oplegnathus fasciatus (48% similarity) and Larimichthys crocea (47% similarity). A real-time reverse transcription PCR analysis showed that under normal circumstances, FII was most highly expressed in the liver, followed by the gill, spleen, thymus, and head-kidney (P < 0.001). After injection of the grass carp reovirus 873 (GCRV873), the pattern of FII expression was significantly altered (P < 0.001); gene expression was high after injection, suggesting a response involving the initiation of the coagulation system and defense of the body in combination with the platelet and complement system.

[Evaluation of lower facial esthetics in females with different skeletal patterns].

Objective: To evaluate lower facial profile in females in different skeletal patterns. Methods: Investigation pictures of three females with beautiful lower facial profiles from Department of Orthodontics, Henan Stomatological Hospital were collected. The skeletal patterns of these females were classified as average, low and high angle, respectively.Upper lip process point (UL) was moved backwards horizontally to reach towards the E line and go even further gradually in above pictures. The distance changed according to E line was defined as DE value. If UL was in front of E line,DE value was denoted as positive, or else negative. Collectively, we obtained 30 pictures (10 pictures in each skeletal facial type) with different DE values (-5, -4, -3, -2, -1, 0, 1, 2, 3, 4 mm), which were divided into average, low and high angle group according to the skeletal facial type. The pictures were evaluated by 144 randomly-selected adult orthodontic patients [66 males, 78 females, aged (29.4±7.7) years] who visited Department of Orthodontics, Henan Stomatological Hospital from June to September, 2019 and 138 orthodontists (including qualified orthodontists and postgraduate orthodontic students [60 males, 78 females, aged (32.2±7.1) years] who participated orthodontics conferences in Henan Stomatological Hospital in June, 2019. The acceptance rate was calculated and rate above 60% was deemed as acceptable DE range. Evaluators were also asked to choose the most esthetic profiles for the best DE value in each skeletal facial type.Data discrepancy was analyzed using Kruskal-Wallis analysis and chi-square test. Results: Most accepted DE was -2 mm among total investigators including orthodonticpatients and orthodontists. There was no difference in total acceptance rate between orthodontists and patients (P>0.05). There was statistic difference in total acceptance rate in different skeletal patterns between orthodontic patients and orthodontists (P<0.05). In total investigators, total acceptance rate was 62.1% (1 752/2 820) in average angle group, 55.4%(1 563/2 820) in high angle group and 33.5%(946/2 820) in low angle group, respectively. Acceptable DE range in three facial types was -4~2 mm (average angle), -2~2 mm (high angle) and -2~-1 mm (low angle), respectively. Conclusions: According to the evaluation of both orthodontic patients and orthodontists, the best DE was-2 mm.Total acceptance rate and acceptable DE range ranked first in average angle group, second in high angle group and third in low angle group.

Minocycline enhances inhibitory transmission to substantia gelatinosa neurons of the rat spinal dorsal horn.

Minocycline, a second-generation tetracycline, is well known for its antibiotic, anti-inflammatory, and antinociceptive effects. Modulation of synaptic transmission is one of the analgesic mechanisms of minocycline. Although it has been reported that minocycline may suppress excitatory glutamatergic synaptic transmission, it remains unclear whether it could affect inhibitory synaptic transmission, which also plays a key role in modulating pain signaling. To examine the effect of minocycline on synaptic transmission in rat spinal substantia gelatinosa (SG) neurons, we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) using whole-cell patch-clamp recording at a holding potential of 0 mV. Bath application of minocycline significantly increased the frequency but not the amplitude of sIPSCs in a reversible and concentration-dependent manner with an EC50 of 85. The enhancement of inhibitory synaptic transmission produced by minocycline was not affected by the glutamate receptor antagonists CNQX and D-APV or by the voltage-gated sodium channel blocker tetrodotoxin (TTX). Moreover, the potency of minocycline for facilitating sIPSC frequency was the same in both glycinergic and GABAergic sIPSCs without changing their decay phases. However, the facilitatory effect of minocycline on sIPSCs was eliminated in a Ca(2+)-free Krebs solution or by co-administration with calcium channel blockers. In summary, our data demonstrate that baseline inhibitory synaptic transmission in SG neurons is markedly enhanced by minocycline. This may function to decrease the excitability of SG neurons, thus leading to a modulation of nociceptive transmission.

Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification.

AIM: To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limited histopathological samples. METHODS: Trace amounts of genomic DNA extracted from fresh tissue and individual lymphoid follicles microdissected from archival paraffin wax tissue sections were amplified using a two-phase PCR protocol with random hexamers as primers (RP-PCR). The randomly amplified DNA samples were used as templates for specific PCR amplifications. To check the fidelity of the RP-PCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing. RESULTS: Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including those for beta globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7-9), and rearranged immunoglobulin heavy chain gene fragments (VH framework 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specific PCR reactions by SSCP or sequencing when compared with the products from the original DNA. CONCLUSION: This method is simple and reliable, and permits multiple genetic analyses when only a limited amount of tissue is available.

Correlations of blood lipids and lipoproteins with coronary artery disease documented by angiography.

A study of the relationships between serum TC, TG and HDL.C and selective coronary arteriography was carried out on 117 patients who were divided into groups according to the extent of artery stenosis. Normal control group consisted of 153 healthy subjects. There were no statistical differences in TC, TG, HDL.C, LDL.C, TC/HDL.C, LDL.C/HDL.C and TC-HDL.C/HDL.C between normal control group and the group with normal coronary arteriogram. LDL.C, TC/HDL.C, LDL.C/HDL.C, TC HDL.C/HDL.C rose and HDL.C decreased as the degree of coronary artery stenosis and the extent of stenosis increased, besides the medium and severe stenosis group. Analyses based on the correlation coefficients indicate that 3 compound indexes (TC-HDL.C/HDL.C, TC-HDL.C, LDL.C/HDL.C) are better in assessing CAD than single index such as LDL.C, HDL.C and TC. The results of our study showed that the 3 compound indexes might be regarded as important risk factors for CAD.

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction.

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.

Nodular lymphocyte predominance Hodgkin's disease: a monoclonal or polyclonal B-cell disorder?

Nodular lymphocyte predominance Hodgkin's disease (NLPHD) is characterized by the presence of atypical putatively neoplastic cells (L & H cells) with a B-cell phenotype. A proportion of patients with NLPHD develop a simultaneous or subsequent large cell B lymphoma (LCL) that is thought to evolve directly from the L & H cells of NLPHD. However, the clonal nature of L & H cells remains controversial, and the relationship between NLPHD and complicating LCL has not been fully established. In an attempt to determine the clonality of L & H cells and to clarify the link between NLPHD and complicating LCL, we used polymerase chain reaction (PCR) to analyze 33 cases of NLPHD, including 15 cases with simultaneous or subsequent LCL, for clonal immunoglobulin (lg) heavy chain variable region (VH) gene rearrangements. PCR amplifications with consensus primers covering framework 2 or framework 3 to joining region were performed on paraffin-embedded tissue sections and, in 12 cases, on microdissection-enriched L & H cells. No clonal Ig rearrangements were detected. In eight of the 15 LCL, monoclonal IgVH regions were amplified, four of which were cloned and sequenced. Clone specific primers were designed based on the unique N region sequences. These allowed detection of LCL clones at a sensitivity up to 1,000 times greater than the consensus primers, as determined by dilution assays. However, no LCL clones were detected in the preceding NLPHD, including microdissection-enriched L & H cells. Our results suggest that populations of L & H cells do not carry monoclonal Ig rearrangements and provide no evidence for a clonal link between NLPHD and complicating LCL.

Nonimmunoglobulin gene hypermutation in germinal center B cells.

Somatic hypermutation is the most critical mechanism underlying the diversification of Ig genes. Although mutation occurs specifically in B cells during the germinal center reaction, it remains a matter of debate whether the mutation machinery also targets non-Ig genes. We have studied mutations in the 5' noncoding region of the Bcl6 gene in different subtypes of lymphomas. We found frequent hypermutation in follicular lymphoma (25 of 59 = 42%) (germinal center cell origin) and mucosa-associated lymphoid tissue (MALT) lymphoma (19 of 45 = 42%) (postgerminal center), but only occasionally in mantle cell lymphoma (1 of 21 = 4.8%) (pregerminal center). Most mutations were outside the motifs potentially important for transcription, suggesting they were not important in lymphomagenesis but may, like Ig mutation, represent an inherent feature of the lymphoma precursor cells. Therefore, we investigated their normal cell counterparts microdissected from a reactive tonsil. Bcl6 mutation was found in 13 of 24 (54%) clones from the germinal centre but only in 1 of 24 (4%) clones from the naive B cells of the mantle zone. The frequency, distribution, and nature of these mutations were similar to those resulting from the Ig hypermutation process. The results show unequivocal evidence of non-Ig gene hypermutation in germinal center B cells and provide fresh insights into the process of hypermutation and lymphomagenesis.

Parvovirus B19 is associated with benign testes as well as testicular germ cell tumours.

AIMS: Parvovirus B19 has been demonstrated in testes of patients with germ cell tumours but not in controls, raising the possibility that the virus has an aetiological role in these tumours. The aims of this study were to investigate the association of the virus with germ cell tumours and to localise the virus histologically. METHODS: DNA was extracted from paraffin wax embedded sections of testes from 10 seminomas, eight teratomas, two mixed seminoma/teratomas, and 10 testes showing benign histology. Polymerase chain reaction (PCR) amplification of three regions within the NS and VP1/2 genes was carried out in duplicate on all samples. One PCR positive case (seminoma/teratoma) was examined by microdissection of histologically defined tissue components followed by PCR amplification of parvoviral sequences. Samples from PCR positive patients were immunostained using a B19 specific monoclonal antibody. RESULTS: Seven cases were PCR positive, these comprised two of 10 seminomas, one of two mixed tumours, none of eight teratomas, and four of 10 benign controls. PCR analysis of the material microdissected from the seminoma/teratoma showed the presence of the virus in regions of seminoma, teratoma, intratubular germ cell neoplasia, normal tubules, and connective tissue. All patient samples studied immunohistochemically were negative. CONCLUSIONS: This confirms the presence of parvovirus B19 in a proportion of germ cell tumours; however, in one patient, the virus was widespread in the tissue components and not confined to tumour cells. In addition, the virus was present in control benign testes. These data suggest that B19 might not be of aetiological importance in germ cell tumours of testis.

Intestinal dissemination of gastric mucosa-associated lymphoid tissue lymphoma.

Despite increasing identification of concurrent gastric and intestinal lymphomas of mucosa-associated lymphoid tissue (MALT), the clonal relationship between the two tumors and their sequential development are poorly understood. It is also unknown whether the development of these concurrent tumors is closely associated with direct antigen stimulation, which is thought to play an important role in the clonal expansion of low-grade MALT lymphomas. To investigate these, we have studied six cases of concurrent gastric and intestinal MALT lymphomas by polymerase chain reaction (PCR) amplification, cloning, and sequencing of the rearranged Ig gene, a strategy that has been widely used for analysis of clonality and antigen-driven properties of B-cell malignancies. In each case, an identical or nearly identical complementarity determining region (CDR) 3 sequence was observed between the dominant clones of concurrent gastric and intestinal MALT lymphomas. In four of six cases examined, sufficient Ig variable region sequence information was obtained to permit analysis of somatic mutations. The mutation patterns in one case suggest that the intestinal lesion is secondary to the gastric tumor, and the mutation patterns in two cases indicate that the gastric and intestinal lesions are derived from different tumour subclones, which emerge after expansion of a common early tumor clone. Furthermore, three of four cases showed ongoing Ig mutations among different PCR clones at each site. These results show that concurrent gastric and intestinal MALT lymphomas are derived from the same clone and suggest that the intestinal lesions result from dissemination of gastric tumours. Antigen stimulation may play a role in tumor evolution, particularly at an early stage.

Simultaneous phenotypically distinct but clonally identical mucosa-associated lymphoid tissue and follicular lymphoma in a patient with Sjögren's syndrome.

A 44-year-old woman with a 12-year history of Sjögren's syndrome (SS) developed a low-grade mucosa-associated lymphoid tissue (MALT) lymphoma in the parotid gland. Two years later, she presented with generalized lymphadenopathy and hepatosplenomegaly and a follicular lymphoma was diagnosed. To investigate the relationship of the two histologically distinct lymphomas, we re-examined their histology and immunophenotype and studied the lymphomatous tissue from the parotid, cervical lymph node, and spleen using molecular genetic methods. Histologic and immunophenotypic studies confirmed the previous diagnoses and also identified a previously unnoticed focus of follicular lymphoma in the second parotid gland biopsy. Polymerase chain reaction (PCR) amplification of the rearranged Ig heavy-chain gene showed the same sized dominant product in the MALT lymphoma and the follicular lymphoma. Similarly, PCR analysis of the t(14:18) translocation yielded an identical sized band from both MALT and follicular lymphoma. Cloning and sequencing of the Ig PCR products showed an identical CDR3 sequence from each lesion, indicating a common clonal lineage. The follicular lymphoma of the parotid gland lymph node and the follicular lymphoma of the spleen showed an identical mutation signature to that of the salivary gland MALT lymphoma. We propose that follicular lymphoma in the parotid gland lymph node may have resulted from colonization of lymphoid follicles by MALT lymphoma cells, following which the tumor cells were induced to express a follicular lymphoma phenotype, due to Bcl-2 overexpression caused by t(14;18), leading to a change in clinical behavior resulting in rapid widespread dissemination of disease. These observations suggest that the distinct phenotypes of low-grade B-cell lymphomas may be the consequence of interplay between genetic and local microenvironmental factors.

Preferential dissemination of B-cell gastric mucosa-associated lymphoid tissue (MALT) lymphoma to the splenic marginal zone.

The tendency for gastric mucosa-associated lymphoid tissue (MALT) lymphoma cells preferentially to localize around reactive B-cell follicles, both in the mucosa and regional lymph nodes, coupled with their immunophenotype, has led to the proposal that the normal cell counterpart of this lymphoma is the marginal zone B cell. In keeping with this proposition, lymphocytes expressing the lymphoma idiotype have been detected in the splenic marginal zone in a single case of gastric MALT lymphoma. To confirm that this truly represented preferential homing of MALT lymphoma to the splenic marginal zone, we have now re-examined this case, together with 17 other cases, using both immunohistochemical and molecular methods in an attempt to establish clonal identity between the gastric lymphoma and cells in the splenic marginal zone. In three cases, the spleen was characterized by marked expansion of marginal zones by cells showing the same pattern of Ig light chain restriction as the gastric lymphoma. None of the remaining 15 cases showed histologic evidence of lymphomatous infiltration. Analysis of the Ig genes by polymerase chain reaction (PCR), cloning, and sequencing confirmed clonal identity between the splenic marginal zone infiltrates and the gastric lymphoma in the histologically involved cases. Amplifiable DNA could be extracted from only 5 of the remaining 15 cases. In 3 of these cases, including the case previously studied using an anti-idiotype, involvement of the splenic marginal zone could be confirmed using microdissection and clone-specific PCR. No involvement could be detected in the remaining 2 cases. In addition, we have shown that mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the primary homing receptor of gut-mucosa for lymphocytes, was strongly expressed by the sinus lining cells of the splenic marginal zone. These results provide strong evidence for preferential involvement of the marginal zone when gastric MALT lymphomas disseminate to the spleen, which is in keeping with the notion that the marginal zone B cells are the normal counterparts of MALT lymphoma cells.