RESUMO
PURPOSE: Remimazolam is a new ultra-short-acting benzodiazepine with unknown effects on cerebral circulation. We measured total cerebral hemoglobin concentrations, which reflect cerebral blood volume (CBV), and cerebral oxygen saturation, using time-domain near-infrared spectroscopy, which can measure the absolute values of cerebral hemoglobin concentrations. We also measured cerebral blood flow velocity (CBFV) in the middle cerebral artery using transcranial Doppler as an indicator of cerebral blood flow (CBF). We did so to examine the effect of remimazolam on cerebral circulation in humans, as assessed CBV, CBF, and cerebral oxygen saturation. METHODS: This was a prospective, observational study. Fifteen patients without serious complications scheduled for general anesthesia were recruited. We measured total cerebral hemoglobin concentrations, CBFV, and cerebral oxygen saturation throughout the anesthetic induction course with remimazolam. RESULTS: Total cerebral hemoglobin concentrations did not change during the process (p = 0.51). In contrast, the mean CBFV was reduced by 11% (significant, p = 0.04). The drop in mean blood pressure following the induction of anesthesia was 17%; however, it was within the range of cerebrovascular autoregulation. Moreover, cerebral oxygen saturation increased by 4% (statistically significant, p < 0.01). CONCLUSIONS: We found that anesthetic induction with remimazolam did not alter CBV and reduced CBF in uncomplicated patients.
Assuntos
Anestesia , Anestésicos , Humanos , Estudos Prospectivos , Benzodiazepinas/farmacologia , Circulação Cerebrovascular , Hemoglobinas , Anestésicos/farmacologiaRESUMO
PURPOSE: Inflammation after stent graft surgery is known as postimplantation syndrome (PIS) and it causes leukocytosis. However, we have experienced leukopenia in the very early postoperative phase of endovascular surgery at our institution. We investigated leukopenia, an under-recognized phenomenon that occurred after transcatheter aortic valve implantation (TAVI), endovascular aortic repair (EVAR), and thoracic endovascular aortic repair (TEVAR). METHODS: Records of patients who underwent TAVI, EVAR, and TEVAR between March 2018 and February 2019 were retrospectively reviewed. Primary outcomes were the decline rate of white blood cell count (DR-WBC) in the immediate postoperative period and its differences among surgical procedures. The secondary endpoint was the relationship between DR-WBC and infectious complications. Furthermore, the incidence of PIS and its differences among the procedures and associations with DR-WBC were evaluated. RESULTS: A total of 108 patients (TAVI 41, EVAR 37, TEVAR 30) were included. DR-WBC immediately after surgery was higher in the TAVI group when compared with other groups (TAVI, 43.1 ± 22.6%; EVAR, 27.6 ± 17.3%; TEVAR, 25.4 ± 27.4%; P < 0.01). DR-WBC was not significantly different regardless of postoperative infection (P = 0.45) or PIS (P = 0.62). The incidence rate of PIS was higher in the EVAR group compared with the TAVI group, and was not associated with DR-WBC. CONCLUSIONS: Leukopenia was a common phenomenon immediately after endovascular surgery, especially TAVI. It resolved a day after surgery and was not associated with PIS or infectious complications. Therefore, it seems to be a transient abnormal hematological finding and a self-limiting condition.
Assuntos
Aneurisma da Aorta Abdominal , Implante de Prótese Vascular , Procedimentos Endovasculares , Leucopenia , Aneurisma da Aorta Abdominal/cirurgia , Prótese Vascular/efeitos adversos , Implante de Prótese Vascular/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Humanos , Leucopenia/complicações , Leucopenia/etiologia , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Fatores de Risco , Stents/efeitos adversos , Resultado do TratamentoRESUMO
Ethidium monoazide and propidium monoazide (EMA and PMA) have been used in combination with PCR for more than a decade to facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving lower limits of detection and quantification of targeted live cells, fewer laborious procedures, lower costs, and potentially higher-throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds. Palladium compounds can penetrate dead (compromised) but not live bacteria and can be chelated primarily by chromosomal DNA and cell wall transmembrane proteins, with small amounts of DNA-binding proteins in vivo The new mechanism for palladium compounds is obviously different from that of platinum compounds, which primarily target DNA. Combining palladium compounds with PCR (Pd-PCR) in water resulted in discrimination between live and dead Enterobacteriaceae bacteria that was much clearer than that seen with the PMA method. Pd-PCR correlated with reference plating or with the currently used PMA-PCR method for pasteurized milk, based on EN ISO 16140:2003 validation. Pd-PCR enabled us to specifically detect and assay viable Enterobacteriaceae cells at concentrations of 5 to 10 CFU/ml in milk while following U.S./EU regulations after a 4.5-h process in a typical laboratory exposed to natural or electric light, as specified by U.S./EU regulations.IMPORTANCE Ethidium monoazide and propidium monoazide (EMA and PMA) facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving fewer laborious procedures, lower costs, and potentially higher-throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds, which have also a novel reaction mechanism different from that of platinum compounds. In view of testing cost, palladium compounds are also very useful here compared with platinum compounds. Ultimately, the innovative Pd-PCR method may be also substituted for the currently used reference plating methods.
RESUMO
PCR cannot distinguish live microorganisms from dead ones. To circumvent this disadvantage, ethidium/propidium-monoazide (EMA/PMA) and psoralen to discriminate live from dead bacteria have been used for 2 decades. These methods require the use of numerous laborious procedures. We introduce an innovative method that uses platinum compounds, which are primarily used as catalysts in organic chemistry and partly used as anti-cancer drugs. Microorganisms are briefly exposed to platinum compounds in vivo, and these compounds penetrate dead (compromised) microorganisms but not live ones and are chelated by chromosomal DNA. The use of platinum compounds permits clear discrimination between live and dead microorganisms in water and milk (including Cronobacter sakazakii and Escherichia coli) via PCR compared with typically used PMA. This platinum-PCR method could enable the specific detection of viable coliforms in milk at a concentration of 5-10 CFU mL(-1) specified by EU/USA regulations after a 4-h process. For sample components, environmental water contains lower levels of PCR inhibitors than milk does, and milk is similar to infant formula, skim milk and blood; thus, the use of the platinum-PCR method could also prevent food poisoning due to the presence of C. sakazakii in dairy products. This method could provide outstanding rapidity for use in environmental/food/clinical tests. Platinum-PCR could also be a substitute for the typical culture-based methods currently used.
Assuntos
Viabilidade Microbiana , Compostos de Platina/metabolismo , Reação em Cadeia da Polimerase/métodos , Animais , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/genética , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Leite/microbiologia , Sensibilidade e Especificidade , Fatores de Tempo , Microbiologia da ÁguaRESUMO
BACKGROUND: Physical function and knee kinematics recovery after discoid lateral meniscus (DLM) tear surgery are essential for a better prognosis. However, these alterations remain unclear. Therefore, this study aimed to investigate changes in physical function and knee kinematics following saucerization and DLM tear repair. METHODS: We enrolled 16 patients who underwent saucerization and DLM tear repair. Postoperative changes in knee kinematics during gait, and physical function, were evaluated at 3, 6, and 12 months. RESULTS: The peak flexion angle of the operated limb during weight acceptance was significantly higher than that of the contralateral limb at 3 (operated limb: 34.6 ± 8.9°, contralateral limb: 23.7 ± 8.3°; P < 0.01) and 6 months (operated limb: 32.1 ± 9.7°, contralateral limb: 24.6 ± 8.2°; P = 0.03) postoperatively, but not at 12 months (operated limb: 27.1 ± 7.1°, contralateral limb: 23.1 ± 9.5°; P = 0.22) postoperatively. The knee extensor strength of the operated limb was significantly lower than that of the contralateral limb at 3 (operated limb: 1.00 ± 0.59 Nm/kg, contralateral limb: 1.37 ± 0.59 Nm/kg; P = 0.01), 6 (operated limb: 1.22 ± 0.55 Nm/kg, contralateral limb: 1.48 ± 0.60 Nm/kg; P < 0.01), and 12 months (operated limb: 1.39 ± 0.57 Nm/kg, contralateral limb: 1.55 ± 0.64 Nm/kg; P = 0.04) postoperatively. CONCLUSION: Knee extension deficits and extensor weakness persisted at 6 months after saucerization and repair of DLM tears. Postoperative rehabilitation should be focused on knee extension function.
Assuntos
Joelho , Recuperação de Função Fisiológica , Lesões do Menisco Tibial , Lesões do Menisco Tibial/cirurgia , Joelho/fisiopatologia , Marcha , Debilidade Muscular/etiologia , Período Pós-Operatório , Humanos , Masculino , Feminino , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007). METHODS: We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria. RESULTS: For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics. CONCLUSIONS: The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture. GENERAL SIGNIFICANCE: This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.
Assuntos
Antibacterianos/farmacologia , Bacteriemia/tratamento farmacológico , DNA Bacteriano/metabolismo , Citometria de Fluxo/métodos , Listeria monocytogenes/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Marcadores de Afinidade/farmacologia , Azidas/farmacologia , Bacteriemia/metabolismo , Bacteriemia/microbiologia , DNA Bacteriano/genética , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/microbiologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Granulócitos/microbiologia , Humanos , Lactente , Luz , Listeria monocytogenes/crescimento & desenvolvimento , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/microbiologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/microbiologia , Marcadores de FotoafinidadeRESUMO
Slightly acidic electrolyzed water (SAEW) is used as a disinfectant for raw chicken meat. Because its volume for a single immersion exceeds 10 times the weight of meat, a large amount of wastewater is generated. Importantly, a higher frequency of immersion is believed to reduce microbial contamination. The objective of this study was to investigate the effect of SAEW immersion at different frequencies on the disinfection and quality of raw chicken legs, thereby possibly limiting the usage of SAEW. Immersion for 1, 3, and 5 times, with a 7:1 SAEW:meat ratio, and duration of 15 min was tested. Meat quality was evaluated based on total aerobic bacteria, Enterobactericeae, total volatile basic nitrogen, thiobarbituric acid reactive substances, and color. A higher immersion frequency lowered the numbers of total aerobic bacteria and Enterobacteriaceae. Moreover, two immersions with a SAEW:meat ratio of 4:1 and a total immersion time of 6 min reduced the bacterial load as effectively as a single 15-min immersion with a SAEW:meat ratio of 7:1. Higher frequencies of SAEW immersion also resulted in lower total volatile basic nitrogen and lipid oxidation after 0 or 3 days of storage. They did, however, magnify the change in color, resulting in brighter meat. Overall, SAEW treatments with two to five immersions can improve the quality of raw chicken legs and reduce wastewater generation.
RESUMO
Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451 bp) following EMA treatment to completely suppress the amplification of DNA of up to 7 logs (10(7) cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25 U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1 colony forming unit (CFU)/2.22 ml within 7.5 h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1 CFU/2.22 ml).
Assuntos
Contagem de Colônia Microbiana/métodos , Enterobacteriaceae/isolamento & purificação , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , Azidas/metabolismo , DNA Bacteriano/genética , DNA Ribossômico/genética , Enterobacteriaceae/genética , Inibidores Enzimáticos/metabolismo , Etídio/metabolismo , Viabilidade Microbiana , Sensibilidade e EspecificidadeRESUMO
The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.
Assuntos
Azidas/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Azidas/metabolismo , Contagem de Colônia Microbiana , DNA Bacteriano/química , Enterobacteriaceae/citologia , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Microbiologia de Alimentos , Temperatura Alta , Viabilidade Microbiana , RNA Ribossômico 16S/química , RNA Ribossômico 23S/químicaRESUMO
In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.
Assuntos
Azidas/farmacologia , Contagem de Colônia Microbiana/métodos , Microbiologia de Alimentos/métodos , Viabilidade Microbiana , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Bovinos , DNA Bacteriano/genética , Enterobacter/isolamento & purificação , Enterobacteriaceae/isolamento & purificação , Pasteurização , RNA Ribossômico 16S/genética , Sensibilidade e EspecificidadeRESUMO
PURPOSE: Since the development of the anatomic double-bundle anterior cruciate ligament (ACL) reconstruction, many studies have focused on excursion and/or tension of each graft. However, no studies to date have adequately investigated thickness of the graft in anatomic double-bundle ACL reconstruction. To obtain basic knowledge from which an ideal graft thickness can be inferred, thicknesses of the anteromedial bundle (AMB) and posterolateral bundle (PLB) was measured in the normal ACL. METHODS: The right knees of 50 cadavers donated for anatomy instruction were studied. Each ACL was separated into the AMB and PLB, and circumferences at the mid-substance and cross-sectional area at the femoral and tibial footprints were measured in each. RESULTS: Cross-sectional areas of the AMB and PLB were 36 ± 10 and 32.1 ± 10.2 mm² at the femoral footprint, and 60.9 ± 21.8 and 52.2 ± 17.3 mm² at the tibial footprint, respectively. Circumferences at the mid-substance were 14.3 ± 3.3 mm for the ALB and 10.8 ± 3.1 mm for the PLB. A positive correlation was seen between AMB and PLB at each of the three sites. CONCLUSION: The AMB is thicker than the PLB, showing a constant correlation in the normal ACL. This suggests that the anteromedial graft must be thicker than the posterolateral graft at least in actual operations.
Assuntos
Ligamento Cruzado Anterior/anatomia & histologia , Dissecação , Articulação do Joelho/anatomia & histologia , Idoso , Idoso de 80 Anos ou mais , Ligamento Cruzado Anterior/cirurgia , Cadáver , Intervalos de Confiança , Feminino , Fêmur/anatomia & histologia , Fêmur/cirurgia , Humanos , Articulação do Joelho/cirurgia , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Tíbia/anatomia & histologia , Tíbia/cirurgiaRESUMO
The real-time PCR (qPCR) and digital PCR (dPCR) to amplify a single-copy of house-keeping genes (i.e., hsp60, pheS or tuf) are used for the assay of limited microbial species. In general, with a single-copy gene, there are obviously varied DNA sequences for even the same microbial species, which could cause difficulties with design of primers and probes for PCR when targeting various single copy genes. In general, for identification by dPCR (as a representative case: Lactobacillus paracasei), accumulated DNA sequence information of 16S rDNA, which is much more frequently used, should be targeted. In contrast, next-generation sequencing revealed that there are five copies of 16S rDNA in a live L. paracasei MCC1849. Therefore, we aimed to reveal, if heat-killed L. paracasei supplemented in nutritional foods that aid the host immune system have the relevant five copies per chromosomal DNA, and if the relevant copies remain unchanged on the same chromosomal DNA or remain to be different chromosomal DNA fragments. So, we revealed the actual distribution of the potential original five copies of 16S rDNA using our innovative dPCR, in which both 16S rDNA and hsp60 genes were simultaneously elongated. The molecular ratios of 16S rDNA/hsp60 dispersed in the dPCR chip were then estimated. The 16S rDNA/hsp60 molecular ratios of the heat-killed L. paracasei in foods, resultantly ranged from 5.0 to 7.2, being the same or higher than that of the five copies determined by next-generation sequencing. The 16S rDNA copy number/ratio indicated the chromosomal DNA molecular number and the associated cell number. As significance, different nutritional foods could potentially cause the loss of chromosomal DNA of supplemented beneficial microbes to a much greater degree. Our absolute dPCR does not require standard correlative samples for the estimation of final products. The estimation principle of the ratio of 16S rDNA/a house-keeping single-copy gene by our absolute dPCR could lead to a useful and accurate assay for various nutritional foods.
Assuntos
Cromossomos Bacterianos/genética , Lacticaseibacillus paracasei/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Chaperonina 60/genética , Primers do DNA/genética , DNA Bacteriano/genética , Microbiologia de Alimentos , Dosagem de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Temperatura Alta , Lacticaseibacillus paracasei/genética , Análise de Sequência de DNARESUMO
Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 10(8) heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 10(8) heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 10(3) to 10(7) antibiotic-treated L. monocytogenes cells but could detect 10(2) live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 10(3) to 10(7) heat-treated cells but could detect 10(1) live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.
Assuntos
Contagem de Colônia Microbiana/métodos , Listeria monocytogenes/genética , Viabilidade Microbiana , Reação em Cadeia da Polimerase/métodos , Azidas/metabolismo , Toxinas Bacterianas/genética , DNA Topoisomerase IV/antagonistas & inibidores , DNA Bacteriano/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Temperatura Alta , Humanos , Luz , RNA Ribossômico 23S/genética , Sensibilidade e Especificidade , Inibidores da Topoisomerase IIRESUMO
Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.
Assuntos
Cromossomos Bacterianos/genética , Cronobacter/genética , DNA Bacteriano/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Cromossomos Bacterianos/química , Cronobacter/química , DNA Bacteriano/químicaRESUMO
Double-bundle anterior cruciate ligament (ACL) reconstruction using hamstring tendon grafts is a standard procedure for ACL injury. However, its clinical effectiveness is not always satisfactory. One cause of this was problems with the graft-tunnel healing of the posterolateral bundle (PLB) on the femur. To solve this problem, we devised a new anatomic ACL reconstruction technique to improve the graft-tunnel healing of the femoral PLB by using a single-bundle with one bone tunnel on the femoral side and a double-bundle on the tibial side. We have performed 40 procedures with excellent results and no cases of intra- or postoperative complication. This procedure can help improve the graft-tunnel healing around the femoral bone tunnel aperture for the PLB.
Assuntos
Reconstrução do Ligamento Cruzado Anterior/métodos , Humanos , Pessoa de Meia-IdadeRESUMO
There is controversy about the treatment for unstable full radial posterior lateral meniscus tears, particularly that involving the posterior root. Some surgeons have advocated repairing these types of meniscus tears using various techniques, but their methods are somewhat technical. We developed the technique for an all-inside repair for full radial posterior lateral meniscus tears using the Meniscal Viper (Arthrex, Naples, FL). A doubled thread is passed through 1 edge of the radial tear by the Meniscal Viper and is kept in place without tying the knot. The Meniscal Viper is used again to set a new thread, repeating the same procedure to another edge of the tear. At this step, 2 doubled threads are passed through each stump of the tear, and both a loop end and 2 free ends of each thread are located outside of the joint. Then, 2 doubled threads pass the third thread into its own loop, pulling it out. Finally, the third thread becomes the mattress suture over the radial tear site and is fastened by sliding knot techniques. This procedure makes it easy to strictly, smoothly, and less invasively shorten the gap by drawing each stump of the meniscus in the direction of the circumference.
RESUMO
To investigate the direct effect to the cartilage caused by the meniscal repair, we examined patients who underwent an isolated meniscal repair without any other abnormalities by arthroscopic examination. A total of 17 patients were examined by second-look arthroscopy after an average interval of 9 months from the meniscal repair, and have been evaluated the status of the repaired meniscus and of the relative femoral condylar cartilage. Changes in the severity of the cartilage lesion between at the time of meniscal repair and the time of the second-look arthroscopy were considered based on the status of the repaired meniscus. Regardless of the healing status of the repair site, it was possible to prevent degeneration in the cartilage in 9 of the 10 patients who demonstrated no degeneration in the meniscal body. Of the 7 patients who demonstrated degeneration in the meniscal body, progression in cartilage degeneration was noted as 1 grade in 2 patients and 2 grades in another 3 patients. Even in those in which stable fusion of the repair site was achieved, the condition of the inner meniscal body was not necessarily maintained favorably in all cases, indicating that degeneration in the meniscal body was a risk factor for cartilage degeneration. It was concluded that recovery could not be expected even at 9 months after the repair if the lesion had already demonstrated degeneration in the meniscal body at the time of repair.
Assuntos
Artroscopia/métodos , Cartilagem/patologia , Traumatismos do Joelho/cirurgia , Meniscos Tibiais/cirurgia , Lesões do Menisco Tibial , Adolescente , Adulto , Criança , Feminino , Humanos , Traumatismos do Joelho/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
We have experienced 3 cases of comparatively rare anteromedial meniscofemoral ligament in which the anterior horn of the medial meniscus was attached to the posterolateral wall of the femoral intercondylar fossa. In 2 of these cases, there was no attachment to the tibia, whereas in the other, it was connected to the lateral meniscus and also firmly to the tibia. In the 2 with no attachment to the tibia, abnormal mobility in the medial meniscus accompanied by flexion and extension of the knee was observed. Degeneration of the intermediate posterior segment and injury of the anterior horn were also observed. This anomaly induces a future meniscus injury, depending on the attachment state to the tibia.
Assuntos
Ligamentos Articulares/cirurgia , Adolescente , Ligamento Cruzado Anterior/fisiologia , Criança , Humanos , Traumatismos do Joelho/patologia , Traumatismos do Joelho/cirurgia , Articulação do Joelho/anormalidades , Articulação do Joelho/cirurgia , Ligamentos Articulares/anormalidades , Ligamentos Articulares/patologia , Imageamento por Ressonância Magnética , Masculino , Meniscos Tibiais/anormalidades , Meniscos Tibiais/cirurgia , Lesões do Menisco TibialRESUMO
We describe the case of fasciitis-like proliferation in the knee joint in a 52-year-old man. The polypoid lesion developed from the synovial joint capsule around the quadriceps tendon and was impinging on the patellofemoral joint. Histologic and immunohistochemical studies revealed a myofibroblastic proliferation similar to nodular fasciitis. Until now, only 3 other cases have been reported in the English and Japanese language literature.
Assuntos
Artroscopia , Fasciite/diagnóstico , Cápsula Articular/patologia , Articulação do Joelho/patologia , Diagnóstico Diferencial , Fasciite/patologia , Fasciite/cirurgia , Fibroblastos/patologia , Tumores de Células Gigantes/diagnóstico , Humanos , Articulação do Joelho/cirurgia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Sinovite Pigmentada Vilonodular/diagnósticoRESUMO
We present a case with an unusual fracture. A 28-year-old man presented a painful and swollen left elbow after falling down. Radiographs revealed combined fractures in the capitellum and the radial head associated with a medial capsular avulsion. Two osteochondral fragments from the capitellum were found at the operation. One was the free fragment revealed on radiographic examination. Another was not revealed before the operation and was found piercing the fracture line of the radial head. These two fragments were removed. The radial head fracture was reduced and was fixed using two Herbert screws. It is very difficult to detect a fragment of the capitellum that impaled the radial head on a plain radiogram before operation. Also, a capsular injury and/or ligamentous injury is often overlooked. This fact should be kept in mind whenever a non-operatively treated radial head fracture fails to respond as expected.