[Analysis of the whole genome traceability and transmission path simulation experiment of the local cluster COVID-19 epidemic].

Objective: To trace and characterize the whole genome of SARS-CoV-2 of confirmed cases in the outbreak of COVID-19 on July 31, 2021 in Henan Province. Method: Genome-wide sequencing and comparative analysis were performed on positive nucleic acid samples of SARS-CoV-2 from 167 local cases related to the epidemic on July 31, 2021, to analyze the consistency and evolution of the whole genome sequence of virus. Results: Through high-throughput sequencing, a total of 106 cases of SARS-CoV-2 whole genome sequences were obtained. The results of genome analysis showed that the whole genome sequences of 106 cases belonged to the VOC/Delta variant strain (B.1.617.2 clade), and the whole genome sequences of 106 cases were shared with the genomes of 3 imported cases from Myanmar admitted to a hospital in Zhengzhou. On the basis of 45 nucleotide sites, 1-5 nucleotide variation sites were added, and the genome sequence was highly homologous. Conclusion: Combined with the comprehensive analysis of viral genomics, transmission path simulation experiments and epidemiology, it is determined that the local new epidemic in Henan Province is caused by imported cases in the nosocomial area, and the spillover has caused localized infection in the community. At the same time, it spills over to some provincial cities and results in localized clustered epidemics.

[Application of cortical bone trajectory screw and sacral alar screw internal fixation for surgical treatment of lumbar adjacent segment degeneration].

Objective: To explore the application of cortical bone trajectory screw (CBTS) and sacral alar screw (SAS) internal fixation in the treatment of lumbar adjacent segment degeneration (ASD) and evaluate its clinical effect. Methods: Data of 24 patients who were diagnosed with ASD and treated by CBTS or SAS in Beijing Chaoyang Hospital were retrospectively reviewed. There were 14 males and 10 females with a mean age of (67.9±8.2) years. The patients were followed-up for (2.6±0.4) years. Perioperative parameters including operation time, intraoperative blood loss and postoperative time on the ground were counted. All patients were followed-up for at least 2 years. Visual analogue scale (VAS) and the Oswestry disability index (ODI) were compared between pre-operation and at the last follow-up. The internal fixation-related complications, pseudarthrosis and adjacent re-degeneration were evaluated in the follow-up. Results: There were 14 proximal ASD patients, 8 distal ASD patients, 1 both ends ASD patient and 1 ASD patient in between the fusion surgeries. Bone mineral density (BMD) T score of the adjacent vertebrae was -1.98±0.91 on average. The ASD patients were re-operated with CBTS and SAS internal fixation technique. A small incision was made in the revision surgery and the original fixation was not completely cut open and removed. The mean operation time was (125±36) min, mean blood loss was (85±33) ml. The postoperative ambulation time was (3.1±1.9) days, and the hospitalization time was (9.0±2.6) days. Before the operation, the average VAS (back pain) score was 5.2±1.0, the average of VAS (leg pain) score was 6.8±1.9 and ODI was 56.6%±12.8%. VAS score was reduced to 1.4±0.6 (waist pain) and 0.9±0.4 (leg pain). ODI was improved to 13.8%±6.3%. All the difference between preoperative and the last follow-up was statically significant (all P<0.01). No internal fixation failure, pseudarthrosis and adjacent re-degeneration were observed in the final follow-up. Conclusion: The application of CBTS and SAS internal fixation techniques in the surgical treatment of lumbar ASD has the advantages of less trauma, faster postoperative recovery, reliable internal fixation, and fewer complications, especially in patients with low bone mineral density.

Drug-induced pyoderma gangrenosum: a model to understand the pathogenesis of pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is a rare autoinflammatory condition in which the alteration of neutrophil function and the innate immune response play key roles in its pathogenesis. Cases of PG have been reported in patients being treated with certain medications, which may help us to understand some of the possible pathways involved in the aetiology of PG. The aim of this review is to review the cases of PG triggered by certain drugs and try to thoroughly understand the pathogenesis of the disease. To accomplish this, a PubMed search was completed using the following words: pyoderma gangrenosum, neutrophilic dermatosis, pathophysiology, drug-induced pyoderma gangrenosum. In total, we found 43 cases of drug-induced PG. Most of them were caused by colony-stimulating factors and small-molecule tyrosine kinase inhibitors. We propose that drugs induce PG through various mechanisms such as dysfunctional neutrophil migration and function, dysregulated inflammatory response, promotion of keratinocyte apoptosis and alteration of epigenetic mechanisms. PG is a rare condition with complex pathophysiology and drug-induced cases are even more scarce; this is the main limitation of this review. Understanding the possible mechanisms of drug-induced PG, via abnormal neutrophil migration and function, abnormal inflammation, keratinocyte apoptosis and alteration of epigenetic mechanisms would help to better understand the pathogenesis of PG and ultimately to optimize targeted therapy.

Comparative cell attachment, cytotoxicity and antibacterial activity of radiopaque dicalcium silicate cement and white-coloured mineral trioxide aggregate.

AIM: To comparatively examine the cell attachment, cytotoxicity, and antibacterial activity of radiopaque dicalcium silicate cement (RDSC) and ProRoot white-coloured mineral trioxide aggregate (WMTA). METHODOLOGY: AlamarBlue was used for real-time and repeated monitoring of MG63 cell attachment on freshly mixed and set cements. The pH changes in the growth medium at different time-points were also measured. Cytotoxicity evaluation was performed according to ISO 10993-5 specifications. The antibacterial activity of the cement specimens was evaluated using Enterococcus faecalis. RESULTS: There were no significant differences (P > 0.05) between the two cements for cell attachment either in the fresh groups or in the set groups at all culture times. Neither freshly mixed group nor set groups had significant pH differences. In the case of cytotoxicity, RDSC was significantly (P < 0.05) superior to WMTA at 12 and 24 h of incubation. RDSC and WMTA possessed similar antimicrobial activity, substantiated by the formation of growth inhibition zones and bacteriostasis ratio in E. faecalis strains. CONCLUSIONS: The cell attachment, cytotoxicity and antibacterial efficacy of RDSC were comparable to those reported for ProRoot WMTA. The results of the current study suggest that this RDSC could be used as a root-end filling material and root sealer.

Role of the p38 pathway in mineral trioxide aggregate-induced cell viability and angiogenesis-related proteins of dental pulp cell in vitro.

AIM: To investigate the influence of mineral trioxide aggregate (MTA) on angiogenesis of primary human dental pulp cells (hDPCs) via the MAPK pathway, in particular p38. METHODOLOGY: Human dental pulp cells were cultured with MTA to angiogenesis, after which cell viability, ion concentration, osmolality, NO secretion, the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression were examined. PrestoBlue(®) was used for evaluating the proliferation of hDPCs. An enzyme-linked immunosorbent assay was employed to determine vWF and Ang-1 protein secretion in hDPCs cultured on MTA and the control. Cells cultured on the tissue culture plate without the cement were used as the control. The t-test was used to evaluate the significance of the differences between the mean values. RESULTS: Mineral trioxide aggregate elicited a significant (P < 0.05) increased viability compared with the control (15%, 16% and 13% on days 1, 3 and 5 of cell seeding, respectively). MTA consumed calcium and phosphate ions, and released more Si ions in the medium. MTA significantly (P < 0.05) increased the osmolality of the medium to 313, 328 and 341 mOsm kg(-1) after 1, 3 and 5 days, respectively. P38 was activated through phosphorylation, and the phosphorylation kinase was investigated in the cell system after being cultured with MTA. Expression levels for Ang-1 and vWF in hDPCs on MTA were higher than those of the MTA + p38 inhibitor (SB203580) group (P < 0.05) at all of the time-points. CONCLUSIONS: Mineral trioxide aggregate was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si increased the osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signalling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion decreased. These findings verify that the p38 pathway plays a key role in regulating the angiogenic behaviour of hDPCs cultured on MTA.

Computed tomography analysis of knee pose and geometry before and after total knee arthroplasty.

Using a three-dimensional (3D) modality to image patients' knees before and after total knee arthroplasty (TKA) allows researchers and clinicians to evaluate causes of pain after TKA, differences in implant design, and changes in the articular geometry as a result of surgery. Computed tomography (CT) has not been fully utilized to date for evaluating the knee after TKA due to metal artifacts obscuring part of the image. We describe an accurate, validated protocol, which has been implemented in vivo, that improves visibility of the patellofemoral joint, matches implant models automatically in 3D, segments preoperative bone semi-automatically, detects and sets coordinate systems automatically, determines the six degrees of freedom of knee pose and geometry, and allows for multiple other measurements that are clinically relevant. Subjects are imaged at 0° and 30° knee flexion, while pushing on a custom-made knee rig to provide partial loadbearing. With some modifications, the protocol can be adopted by any group with access to a CT scanner and image analysis software, allowing for the investigation of numerous clinical and biomechanical questions.

Suspended aggregates as an immobilization mode for high-density perfusion culture of HEK 293 cells in a stirred tank bioreactor.

Cells of the human embryonic kidney cell line (HEK 293) grown in repeated suspension and perfusion systems were characterized and described. Cell aggregates that formed immediately after the HEK 293 cells were inoculated in stirred vessels in serum-containing Dulbecco's modified Eagle's medium (D-MEM)/F-12 medium. The mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 63 to 239 mum after 1 and 8 days of culture in spinner flasks, respectively. No significant differences in cell performance were observed between HEK 293 cell populations grown as suspended aggregates and those grown as anchored monolayers. Replacing the D-MEM/F-12 with CD 293 medium caused the compact spherical cell aggregates to dissociate into single cells and small irregular aggregates without any apparent effect on cell performance. Moreover, the spherical cell aggregates could reform from individual cells and small aggregates when exposed to the serum-containing D-MEM/F-12 dominant medium. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5-l stirred tank bioreactor for 17 days resulted in a maximum viable cell density of 1.2 x 10(7) cells ml(-1). These results demonstrate the feasibility and proof-of-concept for using aggregates as an immobilization system in large-scale stirred bioreactors because a small-scale culture can be used as easily as the inoculum for larger bioreactors.

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation.

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.

Aneuploid marker assignment in hexaploid oat with the C genome as a reference for determining remnant homoeology.

Nullisomic lines of hexaploid oat Avena sativa L. (2n = 6x - 2 = 40, AACCDD) cultivar Sun II were used to assign 134 DNA sequences to 10 chromosome-associated syntenic groups. A limited set of ditelosomic lines allowed localization of subsets of these sequences to six chromosome arms. Advantages of using such aneuploids in mapping are in the assignment of gene families, monomorphic RFLP sequences, and oat linkage groups to chromosomes. The published hexaploid oat RFLP linkage map has 38 linkage groups, 17 more than expected on the basis of the haploid chromosome number. Using nullisomics, eight linkage groups were assigned to five physical chromosomes; using ditelosomics, three of these linkage groups were assigned to their respective chromosome arms. The A- and D-genome chromosome sets of oat are indistinguishable from each other based on different staining and genomic in situ hybridization techniques, while C-genome chromosomes are distinct. Because chromosomal rearrangements such as translocations and inversions have played an important role in the evolution of hexaploid oat, the distinction of C-genome chromosomes can be used to determine remnant homoeologous segments that exist in the other two genomes. Among the 10 syntenic groups identified, six chromosomes showed sequence homoeology believed to represent segmental homoeologous regions. Owing to various evolutionary forces, segmental homoeology instead of whole chromosome homoeology appears to best describe the genome organization in hexaploid oat.

Comparison of nuclear proteins of several human tumors and normal cells by two-dimensional gel electrophoresis.

Using isoelectric focusing and sodium dodecyl sulfate two-dimensional gel electrophoresis, 9 M urea-extractable nuclear proteins from four human tumor cells (HeLa, Namalwa, acute myelogenous leukemia, and lymphoma) and four normal human cells (IMR-90, WI-38, liver, and lymphocytes) were compared. Two protein spots, 140/7.7 and 54/6.6, were found in all four tumor cells but not in the four normal cells studied. Two protein spots, 56/6.7 and 56/6.9, were found in all four normal cells but not in any of the tumors studied. None of these proteins was common to those found in the earlier studies on rat tumors (H. Takami et al., Cancer Res., 39: 2096-2105, 1979).

Chronic herpes simplex infection in cutaneous T-cell lymphomas.

Therapeutically unresponsive erosive or ulcerative lesions caused by the herpes simplex virus developed in five patients with advanced cutaneous T-cell lymphoma. The chronic progressive character of the herpetic infection probably was the consequence of immunosuppression resulting from advanced lymphoma or its treatment. Herpes simplex infection should be considered in the differential diagnosis of cutaneous ulcerations in patients with cutaneous T-cell lymphomas.

Two-dimensional gel electrophoresis of nuclear phosphoproteins of Novikoff hepatoma and regenerating liver.

Two-dimensional polyacrylamide gel electrophoresis patterns of 32P-labeled nuclear proteins in Novikoff hepatoma and regenerating liver relatively small number of the many nuclear protein spots were phosphorylated. The major phosphoprotein spots differed in size and shape from the stained protein spots. Seven phosphoproteins, 125/5.8, 125/7.2, 100/5.9, 85/5.9, 56/5.1, and 50/5.1 (mol. wt. x 10-(3)/pI in the Novikoff hepatoma nuclei were not found in the 18-h regenerating liver nuclei. One nuclear phosphoprotein, 54/6.5, was found in the liver but not in the hepatoma. The four major phosphoproteins, 45/5.3-6.0, 40/5.3-6.0, 35/5.3-6.0, and 25/5.8-6.8 containing about 60% of the total 32Pi of the nuclear proteins, were found in both types of cells. These proteins were of unusual density and shape. Some proteins, such as 125/5.8 and 85/5.9, were enriched in 0.01 M tris extract of the tumor, and proteins 125/7.2 and 56/6.1 were enriched in 0.35 M NaCl extract of the tumor.

Characteristics of infection of B and T lymphocytes from mice after inoculation with cytomegalovirus.

Viremia was produced by inoculating intravenously BALB/c mice with murine cytomegalovirus. Virus was detected in plasma and granulocytes only during the first 8 days after infection. Lymphocyte-associated viremia, detectable by cocultivation on syngeneic or allogeneic fibroblasts, persisted for at least 4 weeks. Eight to 10 days after infection, sonicated lymphocytes had no demonstrable free virus. When whole lymphocytes with no demonstrable free virus were enclosed in a Millipore chamber and placed on a fibroblastic feeder layer, T cells produced free virus but B cells did not. Compared to normal calf serum, specific hyperimmune serum reduced B cell-associated infectious centers by 74% and T cell-associated infectious centers by only 38%. Normal mouse sera reduced by 36% and 30% infectious center production by B cells and T cells, respectively. Lymphocytes enriched with Fc receptor-positive cells produced significantly more infectious centers than receptor-negative cells.

Fiberoptic esophagogastroscopy, brushings, and biopsy in rabbits (Oryctolagus cuniculus).

The esophagus and stomach of rabbits were examined with a pediatric fiberoptic endoscope. The procedure involved general anesthesia and placement of an endotracheal airway followed by endoscopy. Photography, biopsies, and brushings for histology, cytology, and microbiological cultures were accomplished using this procedure. Rabbits were endoscoped and brushed repetitively on successive days without inducing esophagitis.

Isolation and characterization of a cytosol protein (64/7.2) present in large amounts in rapidly growing hepatomas.

Protein 64/7.2 (molecular weight/isoelectric point) is present in the cytosol of several hepatomas including the Novikoff hepatoma and Morris hepatomas 9618A, 7794A, and 3924A, but it is not present in liver or 18-hr regenerating liver. Quantitatively, its concentration was highest in Novikoff hepatoma (150 microgram/g tissue) and Morris hepatoma 3924A (550 microgram/g tissue), which are rapid-growing tumors, less in Morris hepatoma 7794A (72 microgram/g tissue), which is of intermediate growth rate, and least in the slow-growing Morris hepatoma 9618A (25 microgram/g tissue). Protein 64/7.2 was isolated from Novikoff hepatoma ascites cells in high purity as shown by its migration as a single band on one-dimensional acid-urea-polyacrylamide gels and a single spot on two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gels. Its amino acid composition has an acidic to basic amino acid ratio of 1.6. Its amino-terminal amino acid is lysine, and its carboxyl-terminal amino acid is glycine. Interestingly, the amino acid composition is strikingly similar to that of the phosphorylated Novikoff hepatoma chromatin Protein Cg', partially characterized in our laboratory.

Enhancement of murine cytomegalovirus infection during graft-vs.-host reaction.

A mouse model was used for elucidation of the role of a graft-vs-host reaction in promoting cytomegalovirus infection. F1 (DBA/2 X C3H/He) hybrid mice were infected with murine cytomegalovirus. Five weeks later, a graft-vs.-host reaction was produced in the chronically infected animals by the administration of multiple doses of DBA/2 parental splenocytes. Cytomegalovirus was recovered more often from the organs of mice undergoing graft-vs.-host reaction than from those of control animals (P less than 0.001). These results indicate that the graft-vs.-host reaction alone can enhance murine cytomegalovirus infection in a chronically infected host and may help explain the high incidence of cytomegalovirus infection after bone marrow allograft transplantation in man.

Comparative studies on the '5'-cap' and in vitro translational activity of cytoplasmic and nuclear poly A(+) RNA1.

The translational activities of cytoplasmic poly A(+)RNA of normal rat liver and Novikoff hepatoma cells in the wheat germ cell free system were found to be approximately 15-20 times greater than tose of the corresponding nuclear poly A(+) RNA. The translationsl activities were 85 and 62 pmoles 3H-leucine incorporated/micron g cytoglasmic poly A(+) RNA for the liver and tumor respectively and 3-4 pmoles 3H-leucine incoporated/micron g nuclear poly A(+)RNA. Inasmuch as intergity of the '5'-cap' of mRNA is essential for its translational activity, quantitative comparisons were made of its content in these RNA fractions. Of the total 32P incorporated into the tumor cytoplasmic poly A(+) RNA, 0.41% was in the '5'-cap'; in nuclear poly A(+) RNA, the '5'-cap' contained 0.11%. After periodate oxidation and labeling with KB3H4, m7 guanosine, the 5'-terminal nucleoside in both liver and Novikoff hepatoma nuclear poly A(+) RNA contained approximately 20% as much isotope as in the cytoplasmic poly A(+) RNA. These results suggest the lower translational activity of nuclear poly A(+) RNA is partly related to its lower content of the '5'-cap'. Molecular selection of poly A(+) RNA for transport out of the nucleus or further cytoplasmic processing may account for the higher percentage of the '5-cap' and the greater translational activity of the cytoplasmic poly A(+) RNA. During these studies, it was also found that the m7 guanosine of the '5'-cap' was not removed during translation of the mRNA in the wheat germ system; this result suggests that the '5'-cap' may associate with allosteric binding sites of initiation factor(s).

Evidence for coupled synthesis of mRNA for ribosomal proteins and rRNA.

Two-dimensional gel electrophoresis revealed quantitative differences in the 35S-methionine labeled proteins synthesized in vitro in the wheat germ system containing poly A(+) mRNA of normal liver, regenerating liver or Novikoff hepatoma cells. A group of low molecular weight proteins which comigrated with proteins of 40S ribosomal subunits were synthesized in a much greater concentration with the poly A(+) mRNA from the latter two sources. This correlates with increased synthesis of ribosomal proteins in regenerating liver or tumors which is temporally coupled with the increased nucleolar synthesis of rRNA precursors.