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We demonstrate that Blautia coccoides JCM1395 T has the potential to be used for tumor-targeted live bacterial therapeutics. Prior to studying its in vivo biodistribution, a sample preparation method for reliable quantitative analysis of bacteria in biological tissues was required. Gram-positive bacteria have a thick outer layer of peptidoglycans, which hindered the extraction of 16S rRNA genes for colony PCR. We developed the following method to solve the issue; the method we developed is as follows. The homogenates of the isolated tissue were seeded on agar medium, and bacteria were isolated as colonies. Each colony was heat-treated, crushed with glass beads, and further treated with restriction enzymes to cleave DNAs for colony PCR. With this method, Blautia coccoides JCM1395 T and Bacteroides vulgatus JCM5826 T were individually detected from tumors in mice intravenously receiving their mixture. Since this method is very simple and reproducible, and does not involve any genetic modification, it can be applied to exploring a wide range of bacterial species. We especially demonstrate that Blautia coccoides JCM1395 T efficiently proliferate in tumors when intravenously injected into tumor-bearing mice. Furthermore, these bacteria showed minimal innate immunological responses, i.e., elevated serum tumor necrosis factor α and interleukin-6, similar to Bifidobacterium sp., which was previously studied as a therapeutic agent with a small immunostimulating effect.

This study investigated nicotine dependence among Japanese university students who had reached the smoking age (20 years or older) by the time of the coronavirus disease 2019 (COVID-19) pandemic and examined factors that encourage early smoking cessation. Social dependence on nicotine was evaluated using the Kano Total Social Nicotine Dependence Level (KTSND), and physiological dependence was evaluated using the Fagerström Nicotine Dependence Index (FTND). Of the 356 college students who smoked (4.4% of the total), 182 (51.1%) stated that they were not interested in quitting. Furthermore, 124 (68.1%) of those with no interest in quitting smoking were aware that smoking is a high-risk factor for COVID-19, and 58 (31.9%) were unaware. The group not aware of this risk had significantly higher KTSND scores than the group aware of it. The examination of cigarette type that indicated the users of non-conventional cigarette products and dual-user groups scored significantly higher than the cigarette group on FTND items. Overall, the smokers scored above the normal range for social nicotine dependence, suggesting the need to reduce nicotine dependence to encourage college students who continue to smoke to quit smoking.

Saliva and salivary antimicrobial proteins play important roles in the innate immunity, which prevents infections of orally invading bacteria and viruses. In this study, we compared the secretion rates of salivary lactoferrin (Lac) and lysozyme (Lys) in heat-not-burn (HNB) cigarette smokers and non-smokers. The analysis population for this study included 212 members of the fire department, including 32 HNB cigarette smokers, 17 paper cigarette smokers, 14 combined HNB and paper cigarette smokers, and 149 non-smokers. Salivary Lac and Lys concentrations were assessed using enzyme immunoassay. Saliva secretion was significantly lower among HNB cigarette smokers (p < 0.01) than among non-smokers. Accompanying this result, salivary Lac and Lys secretion rates were significantly lower among smokers, particularly HNB cigarette smokers, than among non-smokers (all p < 0.01). Our findings suggest a possible adverse effect of HNB cigarette on the amount of Lac and Lys released into the oral cavity.

Laboratory animals are used for scientific research in various fields. In recent years, there has been a concern that the gut microbiota may differ among laboratory animals, which may yield different results in different laboratories where in-vivo experiments are performed. Our knowledge of the gut microbiota of laboratory-reared common marmosets (Callithrix jacchus) is limited; thus, in this study, we analyzed the daily changes in fecal microbiome composition, individual variations, and effects of the birth facility in healthy female laboratory-reared marmosets, supplied by three vendors. We showed that the marmoset fecal microbiome varied among animals from the same vendor and among animals from different vendors (birth facility), with daily changes of approximately 37%. The fecal microbiome per vendor is characterized by alpha diversity and specific bacteria, with Bifidobacterium for vendor A, Phascolarctobacterium for vendor B, and Megamonas for vendor C. Furthermore, we found that plasma progesterone concentrations and estrous cycles were not correlated with daily fecal microbiome changes. In contrast, animals with an anovulatory cycle lacked Megamonas and Desulfovibrio bacteria compared to normal estrous females. This study suggests that the source of the animal, such as breeding and housing facilities, is important for in-vivo experiments on the marmoset gut microbiota., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to investigate the prediction accuracy of intraocular lens (IOL) power formulas with artificial intelligence (AI) for high myopia. Cases of highly myopic patients (axial length [AL], >26.0 mm) undergoing uncomplicated cataract surgery with at least 1-month follow-up were included. Prediction errors, absolute errors, and percentages of eyes with prediction errors within ±0.25, ±0.50, and ±1.00 diopters (D) were compared using five formulas: Hill-RBF3.0, Kane, Barrett Universal II (BUII), Haigis, and SRK/T. Seventy eyes (mean patient age at surgery, 64.0 ± 9.0 years; mean AL, 27.8 ± 1.3 mm) were included. The prediction errors with the Hill-RBF3.0 and Kane formulas were statistically different from the BUII, Haigis, and SRK/T formulas, whereas there was not a statistically significant difference between those with the Hill-RBF3.0 and Kane. The absolute errors with the Hill-RBF3.0 and Kane formulas were smaller than that with the BUII formula, whereas there was not a statistically significant difference between the other formulas. The percentage within ±0.25 D with the Hill-RBF3.0 formula was larger than that with the BUII formula. The prediction accuracy using AI (Hill-RBF3.0 and Kane) showed excellent prediction accuracy. No significant difference was observed in the prediction accuracy between the Hill-RBF3.0 and Kane formulas., Competing Interests: The authors declare no conflict of interest.

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography-mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.

Four differently substituted chiral Ni(II)-complexes of dehydroalanine Schiff base were prepared and reacted with BrCF 2 COOEt/Cu under the standard reaction conditions. The observed diastereoselectivity was found to depend on the degree and pattern of chlorine substitution for hydrogen in the structure of the dehydroalanine complexes. The unsubstituted complex gave the ratio of diastereomers ( S )(2 S )/( S )(2 R ) of 66/34. On the other hand, introduction of chlorine atoms in the strategic positions on the chiral ligands allowed to achieve a practically attractive diastereoselectivity of (∼98.5/1.5). Diastereomerically pure major product was disassembled to prepare 9-fluorenylmethyloxycarbonyl (Fmoc) derivative of ( S )-4,4-difluoroglutamic acid., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

We evaluated predictive factors for visual outcomes in patients with idiopathic epiretinal membrane (iERM) after pars plana vitrectomy (PPV). Clinical records for 114 eyes (114 patients, mean age: 70.6 years) with iERM treated by PPV between March 2012 and March 2018 were retrospectively reviewed. Overall, the mean postoperative best-corrected visual acuity (BCVA) and central retinal thickness measured by optical coherence tomography improved as early as 1 month after surgery, and further improved until 3 months (P < 0.01). Multiple linear regression analyses adjusted for the preoperative BCVA showed that older age (B, 0.010; 95% confidence interval, 0.003 to 0.016; P = 0.003) and a shorter axial length (AL; B, -0.059; 95% confidence interval, -0.099 to -0.019; P = 0.005) predicted worse postoperative BCVA. The Mann-Whitney U test showed that the postoperative BCVA was worse in eyes with AL < 23.6 mm than in eyes with AL ≥ 23.6 mm (P = 0.037), and in patients aged ≥69 years than in patients aged <69 years (P = 0.024). The findings may help in evaluating surgical indications for each patient to obtain satisfactory outcomes, irrespective of the preoperative BCVA.

Visual field defects interfere with free actions and influence the quality of life of patients with retinitis pigmentosa; the prevalence of this disease is increasing in aging societies. Patients with progressive disease may require visual aids; however, no such devices are currently available. We utilized a retinal projection eyewear system, QD laser eyewear, which includes a projector inside the spectacle frame, to draw the image taken by a connected portable camera with a wide field lens. The images are projected onto the retina using a Maxwellian view optical system, which is not influenced by refractive error or the amount of incident light. Goldmann perimetry and figure recognition tests with the QD laser eyewear showed increased visual field areas and angles, and shortened the time for recognition of the number of figures in a sheet, in a limited visual field model that we developed by using a pin-hole system to simulate the tunnel vision of retinitis pigmentosa in 19 healthy adults. The device supported the quality of vision. Additionally, the visual field defect model used in healthy adults was useful for validating the device in the development stage of the study, to clarify both advantages and future goals for improving the device.

Amino acids participate directly and indirectly in many important biochemical functions in the brain. We focused on one amino acid metabolic enzyme, L-amino acid oxidase (LAO), and investigated the importance of LAO in brain function using LAO1 knockout (KO) mice. Compared to wild-type mice, LAO1 KO mice exhibited impaired fear learning and memory function in a passive avoidance test. This impairment in LAO1 KO mice coincided with significantly reduced hippocampal acetylcholine levels compared to wild-type mice, while treatment with donepezil, a reversible acetylcholine esterase inhibitor, inhibited this reduction. Metabolomic analysis revealed that knocking out LAO1 affected amino acid metabolism (mainly of phenylalanine [Phe]) in the hippocampus. Specifically, Phe levels were elevated in LAO1 KO mice, while phenylpyruvic acid (metabolite of Phe produced largely by LAO) levels were reduced. Moreover, knocking out LAO1 decreased hippocampal mRNA levels of pyruvate kinase, the enzymatic activity of which is known to be inhibited by Phe. Based on our findings, we propose that LAO1 KO mice exhibited impaired fear learning and memory owing to low hippocampal acetylcholine levels. Furthermore, we speculate that hippocampal Phe metabolism is an important physiological mechanism related to glycolysis and may underlie cognitive impairments, including those observed in Alzheimer's disease.

Purpose: To investigate accommodative microfluctuations (AMFs) and visual function in short tear break-up time (BUT)-type dry eye (DE) and non-DE subjects., Methods: This prospective comparative study included 48 volunteers with DE symptoms (mean age 34.8 ± 5.5 years, age range 25-42 years) and 73 without DE symptoms (mean age 30.6 ± 4.7 years, age range 25-42 years). The eyes were divided into two groups: (1) DE group with DE symptoms and BUT ≤ 5 s and (2) non-DE group without DE symptoms and BUT > 5 s. We excluded eyes with Schirmer score ≤ 5 mm and positive keratoconjunctival epithelial damage. Tear evaluation, AMF, and functional visual acuity (VA) examinations were performed. AMF parameters included total high-frequency component (HFC), HFC with low accommodation for the task of staring into the distance (HFC1), and HFC with high accommodation for deskwork (HFC2). Functional VA parameters included starting VA, functional VA, visual maintenance ratio, and blink frequency., Results: A total of 33 and 34 eyes were categorized in the DE and non-DE groups, respectively. Mean blink frequency and HFC1 values were significantly higher in the DE group than they were in the non-DE group., Conclusions: DEs with symptoms showed abnormal AMF and visual function, which may be associated with DE symptoms.

Background: Lutein and zeaxanthin are suggested micronutrient supplements to prevent the progression of age-related macular degeneration (AMD), a leading cause of blindness worldwide. To monitor the levels of lutein/zeaxanthin in the macula, macular pigment optical density (MPOD) is measured. A commercially available device (MPSII®, Elektron Technology, Switzerland), using technology based on heterochromatic flicker photometry, can measure both absolute and estimated values of MPOD. However, whether the estimated value is applicable to Asian individuals and/or AMD patients remains to be determined., Methods: The absolute and estimated values of MPOD were measured using the MPSII® device in 77 participants with a best-corrected visual acuity (BCVA) > 0.099 (logMAR score)., Results: The studied eyes included 17 young (20-29 years) healthy, 26 aged (>50 years) healthy, 18 aged and AMD-fellow, and 16 aged AMD eyes. The mean BCVA among the groups were not significantly different. Both absolute and estimated values were measurable in all eyes of young healthy group. However, absolute values were measurable in only 57.7%, 66.7%, and 43.8%, of the aged healthy, AMD-fellow, and AMD groups, respectively, and 56.7% of the eyes included in the 3 aged groups. In contrast, the estimated value was measurable in 84.6%, 88.9% and 93.8% of the groups, respectively, and 88.3% of eyes in the pooled aged group. The estimated value was correlated with absolute value in individuals from all groups by Spearman's correlation coefficient analyses (young healthy: R 2  = 0.885, P = 0.0001; aged healthy: R 2  = 0.765, P = 0.001; AMD-fellow: R 2  = 0.851, P = 0.0001; and AMD: R 2  = 0.860, P = 0.013). Using the estimated value, significantly lower MPOD values were found in aged AMD-related eyes, which included both AMD-fellow and AMD eyes, compared with aged healthy eyes by Student's t-test (P = 0.02)., Conclusions: Absolute, in contrast to estimated, value was measurable in a limited number of aged participants; however, it was correlated with estimated value both in young and aged Asian populations with or without AMD. These results may inform future clinical studies investigating the measurement of MPOD in understanding the role of macular pigments in the pathogenesis of AMD.

Purpose: To evaluate visual function in patients with early presbyopia using the functional visual acuity (FVA) test., Methods: This study included 27 eyes of 27 healthy older volunteers (mean age, 44.1 ± 2.6 years) and 14 eyes of 14 healthy young volunteers (mean age, 28.4±4.8 years). The distance-corrected visual acuity (DCVA), distance-corrected near VA (DCNVA), subjective amplitude of accommodation (AA), and distance and near pupillary diameters were measured. The distance FVA and distance-corrected near FVA (DCNFVA) were measured using the FVA Measurement System. The standard Schirmer test and standard tear break-up time measurement also were performed., Results: The logarithm of the minimum angle of resolution (logMAR) DCVA was better than 0 in all subjects. The percentages of subjects with logMAR DCNVA below 0 was significantly lower in the presbyopia group than in the young group. The DCNFVA in the presbyopia group was significantly (P < 0.001) poorer than the DCNVA in that group. Significant linear negative correlations were seen between the DCNVA and AA (r = -0.507, P < 0.001) and the DCNFVA and AA (r = -0.681, P < 0.001) in the older subjects. Stepwise regression analysis showed that only the AA was a significant factor predictive of the DCNFVA in the presbyopia group. Tear function parameters were not adopted in the regression model., Conclusions: Measurement of the DCNFVA can detect decreased AA in early presbyopia better than measurement of the conventional near VA. The DCNFVA is a good index for early presbyopia.

Purpose: To investigate the changes in functional visual acuity (VA) and higher order aberrations in dry eye patients., Methods: In this prospective comparative case series, 22 right eyes were classified into those with or without superficial punctate keratopathy (SPK) in the central cornea of 22 patients with Sjögren syndrome; 10 right eyes of 10 normal subjects served as the control. Serial measurements of VAs using a functional VA measurement system and higher order aberrations using a wavefront sensor were performed under blink-free conditions without topical anesthesia over a 10-second period. The parameters for each measurement were compared among the SPK-positive and -negative and normal groups. The correlation between those parameters was also analyzed., Results: Dry eye with SPK showed significant deterioration of visual function and optical quality compared with dry eye without SPK and in normal eyes, as detected by both the visual maintenance ratio (VMR; P < 0.05) and the variation of VA (P < 0.05) and by comalike and total higher order aberrations (P < 0.05). Moreover, the severity of epithelial damage at the central cornea correlated significantly with VMR (P < 0.01) and variation of VA (P < 0.01) as well as comalike (P < 0.05) and total higher order aberrations (P < 0.05). The dry eye group without SPK showed minor visual deterioration compared with normal eyes, as detected only by VMR (P < 0.05)., Conclusions: Optical disturbances at the central optical zone of the cornea in dry eye disease may affect visual performance. Functional VA measurement may be an applicable method of evaluating visual performance in dry eyes that is as efficient as wavefront aberration measurements.

For rapid monomerization of biodegradable plastics, various microorganisms were screened and TB-71 was selected as the best strain. TB-71 degraded solid poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(ethylene succinate), and poly(epsilon-caprolactone) but not poly(butylene succinate), poly(2-hydroxybutylate-co-valerate) or poly(lactic acid). Esterase activity was observed in the culture broth during PBSA degradation, which was specifically induced by PBSA. Analysis of the degradation products revealed that PBSA was degraded to monomers.

Many poly(lactic acid) (PLA)-degrading microorganisms have been isolated from the natural environment by culture-based methods, but there is no study about unculturable PLA-degrading microorganisms. In this study, we constructed a metagenomic library consisting of the DNA extracted from PLA disks buried in compost. We identified three PLA-degrading genes encoding lipase or hydrolase. The purified enzymes degraded not only PLA, but also various aliphatic polyesters, tributyrin, and p-nitrophenyl esters. From their substrate specificities, the PLA depolymerases were classified into an esterase rather than a lipase. Among the PLA depolymerases, PlaM4 exhibited thermophilic properties; that is, it showed the highest activity at 70 degrees C and was stable even after incubation for 1 h at 50 degrees C. PlaM4 had absorption and degradation activities for solid PLA at 60 degrees C, which indicates that the enzyme can effectively degrade PLA in a high-temperature environment. On the other hand, the enzyme classification based on amino acid sequences showed that the other PLA depolymerases, PlaM7 and PlaM9, were not classified into known lipases or esterases. This is the first report on the identification and characterization of PLA depolymerase from a metagenome.

A bacterium which degrades urethane compounds was isolated and identified as Rhodococcus equi strain TB-60. Strain TB-60 degraded toluene-2,4-dicarbamic acid dibutyl ester (TDCB) and accumulated toluene diamine as the degradation product. The enzyme which cleaves urethane bond in TDCB was strongly induced by acetanilide. The purified enzyme (urethane hydrolase) was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 55 kDa. The optimal temperature and pH were 45 degrees C and 5.5, respectively. The enzyme hydrolyzed aliphatic urethane compound as well as aromatic ones. The activity was inhibited by HgCl(2), p-chrolomercuribenzoic acid, and phenylmethylsulfonyl fluoride, suggesting that cysteine and/or serine residues play an important role in the activity. The enzyme catalyzed the hydrolysis of anilides, amides, and esters as well as TDCB. It was characterized as a novel amidase/esterase, differing in some properties from other known amidases/esterases.

Downregulation of oligodendrocyte-related genes, referred to as oligodendrocyte dysfunction, in schizophrenia has been revealed by DNA microarray studies. Because oligodendrocyte-specific transcription factors regulate the differentiation of oligodendrocytes, genes encoding them are prime candidates for oligodendrocyte dysfunction in schizophrenia. We found that the cytosine-guanine dinucleotide (CpG) island of sex-determining region Y-box containing gene 10 (SOX10), an oligodendrocyte-specific transcription factor, tended to be highly methylated in brains of patients with schizophrenia, correlated with reduced expression of SOX10. We also found that DNA methylation status of SOX10 also was associated with other oligodendrocyte gene expressions in schizophrenia. This may be specific to SOX10, because the CpG island of OLIG2, which encodes another oligodendrocyte-specific transcription factor, was rarely methylated in brains, and the methylation status of myelin-associated oligodendrocytic basic protein, which encodes structural protein in oligodendrocytes, did not account for their expressions or other oligodendrocyte gene expressions. Therefore, DNA methylation status of the SOX10 CpG island could be an epigenetic sign of oligodendrocyte dysfunction in schizophrenia.

To establish an environmentally friendly groundwater bioremediation process using a cellulose carrier combined with cellulose-utilizing, denitrifying microorganisms, a novel psychrophilic bacterium, designated CL-5, which can degrade a commercial-based cellulose carrier as the sole carbon source, was screened. Since the denitrification capability of CL-5 is low, complex microbial systems were constructed together with other denitrifying bacteria designated NR-1 and NR-2 that were also isolated from soil. The nitrate-reducing activities of mixed cultures were much higher than those of the pure cultures of CL-5, NR-1 and NR-2. The highest N(2)O and N(2) formation activities were observed in the mixed culture of CL-5+NR-2.

The exact etiology of schizophrenia remains undetermined but accumulating evidence suggests that disturbances in neurodevelopment may represent one contributory factor. Netrin G1, a recently cloned gene from the mouse, has been shown to play a potential role in the formation of neural circuitry. To determine whether this gene is involved in the development of psychosis, we performed a genetic association study of human netrin G1 gene in schizophrenia. First, we determined the human genomic structure of netrin G1 by direct comparisons between cDNA and genome sequences, and by database searches. For the subsequent examination of heterozygosity, we selected 10 single nucleotide polymorphisms (SNPs) for an association test in case (n = 180) and control (n = 180) samples. Among these SNPs, IVS8-1467C>T showed significant allelic association (nominal P = 0.020) with disease. This SNP is located in a haplotype block of approximately 40 kb and haplotypes in this block also displayed significant association (most significant P = 0.017). These findings suggest that netrin G1 or a nearby gene may contribute to the overall genetic risk for schizophrenia.

Pyoverdine-type siderophores from fluorescent Pseudomonas species were purified by Zn2+-chelate chromatography, and their matrix metalloproteinase-2 (MMP-2) inhibition and metal-ion-chelating activities were studied. Structurally different pyoverdines showed different MMP-2 inhibition activities, and the inhibition activity was correlated with Zn2+-chelating activity. The IC50 value of a pyoverdine ((P113A1)-2, MW 1187) for MMP-2 was 0.27 microg/ml (0.23 microM).

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

Transthoracic needle aspiration of the lung in guinea pigs with experimental pseudomonas pneumonia was evaluated. The number of bacteria in aspirates correlated well with that of bacteria in the lungs which showed diffuse pneumonia (10(7) inoculum group). The number of deaths of experimental animals increased together with an increase of the times of aspiration. This procedure was also investigated in 16 patients of pneumonia and 17 patients of pulmonary abscess. The isolation rate of pathogen from pneumonia was 31.3% and that from pulmonary abscess 58.8%. A higher isolation rate was obtained with purulent aspirates. Predominantly anaerobic bacteria were isolated, and in pulmonary abscess usually in association with other bacteria. This method could be applied with success, for determining responsible pathogens. Even the normal oropharyngeal flora such as alpha-Streptococcus could be identified as pathogens. The complication rate was relatively low (6 out of 33 patients, 18.2%) including hemoptysis as a major one and pneumothorax or bloody sputum as minor ones. Transthoracic needle aspiration was reevaluated in experimental and clinical materials and was found to be an excellent and safe method for determining the responsible pathogen of respiratory infection.