SARS-CoV-2 Contamination on Healthy Individuals' Hands in Community Settings During the COVID-19 Pandemic.

Introduction Hand hygiene is an infection control measure for COVID-19 in our daily lives; however, the contamination levels of SARS-CoV-2 in the hands of healthy individuals remain unclear. Thus, we aimed to evaluate SARS-CoV-2 contamination levels by detecting viral RNA and viable viruses in samples obtained from the hands of 925 healthy individuals. Methods Swab samples were collected from the palms and fingers of healthy participants, including office workers, public officers, university students, university faculty and staff, and hospital staff between December 2022 and March 2023. The collected swab samples were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2 RNA detection. Viral RNA-positive samples were subjected to plaque assay to detect viable viruses. Results We collected 1,022 swab samples from the hands of healthy participants. According to the criteria for data collection, 97 samples were excluded, and 925 samples were analyzed using RT-qPCR. SARS-CoV-2 RNA was detected in three of the 925 samples. The viral RNA detection rate was 0.32% (3/925), and the viral RNA copy numbers ranged from 5.0×103 to 1.7×105 copies/mL. The RT-qPCR-positive samples did not contain viable viruses, as confirmed by the plaque assay results. Conclusions The detection rate of SARS-CoV-2 RNA from the hands of healthy individuals was extremely low, and no viable viruses were detected. These results suggest that the risk of contact transmission via hands in a community setting is extremely rare.

Fluctuation in SARS-CoV-2 Environmental Surface Contamination Levels in Homes Where Patients With COVID-19 Stayed for Recuperation.

Introduction Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) often occurs among family members. Elucidating where viable SARS-CoV-2 virions, and not just residual viral RNA, are present in the house is necessary to prevent the further spread of the coronavirus disease 2019 (COVID-19). We aimed to evaluate the environmental surface contamination levels of both SARS-CoV-2 RNA and viable viruses in the homes of housebound patients with COVID-19. Methods Environmental samples were collected from the households of three patients in April and July 2022 when the number of new COVID-19 cases in Japan was reported to be approximately 50,000 and 200,000 cases per day, respectively. For each case, samples were obtained from 19-26 household sites for seven consecutive days. SARS-CoV-2 RNA was examined in 455 samples through reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and RT-qPCR-positive samples were subjected to plaque assay to detect viable viruses. Results Among the 455 samples, 63 (13.8%) that were collected from patients' pillows and comforters, doorknobs, chairs, and refrigerators tested positive by RT-qPCR. The maximum detection rate of SARS-CoV-2 RNA-positive samples in each case ranged from 20.0% to 57.7% on days 1 to 3. The detection rate gradually decreased to 0-5.3% as the days elapsed. Although all RT-qPCR-positive samples were examined, no viable viruses were detected in these samples. Conclusions Although environmental contamination of SARS-CoV-2 RNA was observed in the homes of housebound patients with COVID-19, no viable viruses were isolated. This suggests that the indirect transmission risk from fomites was low.

Levels of environmental contamination with SARS-CoV-2 in hospital rooms and salivary viral loads of patients with coronavirus disease 2019.

BACKGROUND: Clarifying the presence of viable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rather than SARS-CoV-2 viral RNA in inpatient rooms is important for infection control of coronavirus disease 2019 (COVID-19). In this study, we investigated levels of viral RNA and viable virus on environmental surfaces and in patient saliva. METHODS: Environmental samples from 23 sites in hospital rooms were collected every other day until patient discharge. Saliva specimens and samples from the inner surface of patient masks were also collected. Additionally, environmental samples were collected from 46 sites in hospital rooms on discharge day. The samples were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and plaque assays. RESULTS: The 10 enrolled cases were classified as mild COVID-19, and patients were discharged after 6-9 days. The viral RNA was detected in 12.4% (105/849) of serially collected environmental samples during hospitalization, whereas viable virus was detected only in 0.47% (4/849), which were from sinks and tap levers. Although all patients recovered, three cases retained viable virus in the last saliva specimen collected. In the 15 discharged rooms, viral RNA was detected in 6.6% (45/682) of the samples, and viable virus was detected in only one sample from the sink. CONCLUSIONS: Although environmental surfaces surrounding patients with COVID-19 were frequently contaminated with viral RNA, the presence of viable virus was rare and limited only to areas around sinks. These results suggest that contact infection risk via fomites in hospital rooms is extremely rare.

Altered renin-angiotensin system gene expression in airways of antigen-challenged mice: ACE2 downregulation and unexpected increase in angiotensin 1-7.

OBJECTIVE: Evidence suggest that the renin-angiotensin system (RAS) is activated in people with asthma, although its pathophysiological role is unclear. Angiotensin-converting enzyme 2 (ACE2) is the major enzyme that converts angiotensin II to angiotensin 1-7 (Ang-1-7), and is also known as a receptor of SARS-CoV-2. The current study was conducted to identify the change in RAS-related gene expression in airways of a murine asthma model. METHODS: The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. RESULTS: The KEGG pathway analysis of differentially expressed genes in our published microarray data revealed a significant change in the RAS pathway in the antigen-challenged mice. Quantitative RT-PCR analyses showed significant increases in the angiotensin II-generating enzymes (Klk1, Klk1b3 and Klk1b8) and a significant decrease in Ace2. Surprisingly, ELISA analyses revealed a significant increase in Ang-1-7 levels in bronchoalveolar lavage (BAL) fluids of the antigen-challenged animals, while no significant change in angiotensin II was observed. Application of Ang-1-7 to the isolated BSMs had no effect on their isometrical tension. CONCLUSION: The expression of Ace2 was downregulated in the BSMs of OA-challenged mice, while Klk1, Klk1b3 and Klk1b8 were upregulated. Despite the downregulation of ACE2, the level of its enzymatic product, Ang-1-7, was increased in the inflamed airways, suggesting the existence of an unknown ACE2-independent pathway for Ang-1-7 production. The functional role of Ang-1-7 in the airways remains unclear.

Assessment of environmental surface contamination with SARS-CoV-2 in concert halls and banquet rooms in Japan.

BACKGROUND: Although crowds are considered to be a risk factor for SARS-CoV-2 transmission, little is known about the changes in environmental surface contamination with the virus when a large number of people attend an event. In this study, we evaluated the changes in environmental surface contamination with SARS-CoV-2. METHODS: Environmental samples were collected from concert halls and banquet rooms before and after events in February to April 2022 when the 7-day moving average of new COVID-19 cases in Tokyo was reported to be 5000-18000 cases per day. In total, 632 samples were examined for SARS-CoV-2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR) tests, and RT-qPCR-positive samples were subjected to a plaque assay. RESULTS: The SARS-CoV-2 RNA detection rate before and after the events ranged from 0% to 2.6% versus 0%-5.0% in environmental surface samples, respectively. However, no viable viruses were isolated from all RT-qPCR-positive samples by the plaque assay. There was no significant increase in the environmental surface contamination with SARS-CoV-2 after these events. CONCLUSIONS: These findings revealed that indirect contact transmission from environmental fomite does not seem to be of great magnitude in a community setting.

Microvascular Changes in the Cystic Lesion of Branch Retinal Vein Occlusion Imaged by Swept-Source Optical Coherence Tomography Angiography.

Introduction: This study aimed to describe the quantitative features of the microvasculature in the cystic lesions of branch retinal vein occlusion (BRVO). Methods: A total of 43 eyes with BRVO, treated with anti-vascular endothelial growth factor therapy, were analyzed. Using wide-field swept-source optical coherence tomography angiography (OCTA), en face OCT images were obtained by depth-integrated reflectivity of the retina, and vascular density (VD), vascular length (VL), vascular lacunarity, and fractal dimension (FD) were evaluated in a 12 × 12-mm area of retinal nonperfusion. Results: The mean area of affected lesions was 38.7 ± 19.8 mm2, and cystic lesions were 8.5 ± 10.1 mm2. VD, VL, and FD were significantly decreased in the cystic lesions compared to other affected lesions in the same eyes (p = 0.0010, p = 0.0001, and p = 0.0003, respectively) and in all eyes (p = 0.0281, p = 0.0050, and p < 0.0001, respectively). VD in cystic lesions within the vascular arcade (25 eyes) correlated with best-corrected visual acuity on OCTA (r = -0.433, and p = 0.0492). Conclusions: Vascular structure in the cystic lesions was unpreserved compared to the other lesions in BRVO. These findings may help in understanding the pathophysiology of retinal edema in BRVO.

Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma.

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

Umpolung Reaction of α-Imino Thioesters and the Subsequent C-C Bond Formation with the Unexpected Alkylthio Rearrangement.

An umpolung reaction of the α-imino thioester was examined, and we found that α-imino thioesters were more effective substrates for the umpolung N-alkylation than conventional α-imino esters and they gave N-alkylated amino thioesters in high yields under mild reaction conditions in a short time. A new type of C-C bond formation followed by an unexpected rearrangement of the alkylthio group took place with the unsaturated ketones to afford the ß-alkylthio-α-amino thioesters in high yields with good diastereoselectivity.

Isolation and characterization of [5,6]-pyrrolidino-Sc3N@I(h)-C80 diastereomers.

Reactions of Sc3N@I(h)-C80 with aziridine derivatives were conducted to afford the corresponding mono-adducts. A pair of diastereomers of the mono-adduct [5,6]-pyrrolidino-Sc3N@I(h)-C80 was isolated and characterized by means of mass spectrometry, vis-NIR absorption spectroscopy, and electrochemical measurements. Structural analysis of the mono-adducts was conducted by NMR and single-crystal X-ray structure determinations.

Roles of Pseudomonas aeruginosa autoinducers and their degradation products, tetramic acids, in bacterial survival and behavior in ecological niches.

Pseudomonas aeruginosa, an opportunistic pathogen, is known to mainly use N-acylhomoserine lactones (AHLs) as autoinducers. Recent progress in this field demonstrated that not only AHLs, but also their degradation products, tetramic acids, may have some biological activities. The present study examined the roles of Pseudomonas autoinducers and tetramic acids in bacterial survival and behavior in ecological niches. P. aeruginosa autoinducers and the tetramic acid derivatives were chemically synthesized, and the structure-activity correlation was investigated. Some tetramic acids derived from AHLs caused a significant reduction in the viability of P. aeruginosa in a concentration dependent manner (30-300 µM). The smaller the inoculum of bacteria, the stronger the bactericidal activity that was observed. The data from tetramic acid derivatives indicated the keto-enol structure of tetramic acid to be critical for the antibacterial activity. Exogenous tetramic acid did not induce significant changes in the formation of biofilm or production of exoproducts such as pyocyanin and elastase. Tetramic acid and disinfectants acted synergistically to kill P. aeruginosa. These results suggest the AHL-degradation product tetramic acid to be useful for bacterial control.

Evaluation of antimicrobial susceptibility for ß-lactams against clinical isolates from 51 medical centers in Japan (2008).

This antimicrobial resistance surveillance study was performed in 51 medical centers in Japan over an 11-year period. The susceptibilities of 4228 strains including Escherichia coli (491 strains), Klebsiella spp. (462 strains), Enterobacter spp. (459 strains), Citrobacter freundii (358 strains), indole-positive Proteus spp. (386 strains), Serratia spp. (443 strains), Acinetobacter spp. (327 strains), Pseudomonas aeruginosa (473 strains), oxacillin-susceptible Staphylococcus aureus (481 strains), and coagulase-negative staphylococci (CoNS; 348 strains) were tested with 7 ß-lactams (cefepime, cefpirome, ceftazidime, cefoperazone/sulbactam, imipenem, and piperacillin for gram-negative bacteria, or oxacillin for gram-positive bacteria). No resistance to these ß-lactams (with the exception of ceftazidime) was found in oxacillin-susceptible S. aureus and CoNS. Of the E. coli clinical isolates, 24.6% were resistant to piperacillin, whereas 3.5% or less (cefpirome = 4.5%) were resistant to other ß-lactam agents. Klebsiella spp. isolates were more susceptible to imipenem (99.6%), cefepime (98.7%), ceftazidime (98.5%), cefpirome (97.6%), and cefoperazone/sulbactam (97.6%). Isolates of Enterobacter spp., C. freundii, and Serratia spp. were susceptible to imipenem, cefepime, and cefpirome as well. The sensitivities of these organisms against cefepime and cefoperazone/sulbactam were 100%. Acinetobacter spp. isolates were less resistant to cefoperazone/sulbactam (0.6% resistance), imipenem (0.9%), and ceftazidime (2.8%) compared with other ß-lactam antibiotics tested. Isolates of P. aeruginosa were more susceptible to piperacillin (9.1% resistance), cefoperazone/sulbactam (9.5%), and cefepime (6.6%) compared with ceftazidime (10.8%), cefpirome (16.3%), and imipenem (23.5%). The proportion of strains resistant to ß-lactam antimicrobials has decreased compared with data from 2006 (Diagn. Microbiol. Infect. Dis. 60:177-183), reflecting the reduced consumption of ß-lactams in Japan.

Anti-Clostridium difficile potential of tetramic acid derivatives from Pseudomonas aeruginosa quorum-sensing autoinducers.

We have examined the potential bactericidal activities of several tetramic acids derived from Pseudomonas autoinducers against Clostridium difficile, a cause of antibiotic-associated pseudomembranous colitis. Clinical isolates of C. difficile (n=4) were incubated in broth with a chemically synthesized Pseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and the mechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N-3-oxododecanoyl L-homoserine lactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 microM. The outer membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid, because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-staining characteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that the keto-enol structure and the length of the acyl side chain of tetramic acid may be essential for the antibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives to be novel agents with activity against C. difficile.

Contribution of itch-associated scratch behavior to the development of skin lesions in Dermatophagoides farinae-induced dermatitis model in NC/Nga mice.

Recently, we have reported that the pathophysiological features of dermatitis induced by the repeated application with Dermatophagoides farinae (Df) extract ointment in NC/Nga mice were similar to those observed in the patients with atopic dermatitis. In the present study, we first examined whether the application of Df in other mouse strains could induce dermatitis. The repeated application of Df body (Dfb) ointment to the barrier-disrupted back of ICR, C57BL/6, and Balb/c mice did not cause any apparent skin lesions, although transient increase in serum immunoglobulin E (IgE) levels during antigen application was observed. On the other hand, in NC/Nga mice, dermatitis scores and serum IgE levels increased remarkably, and then these changes sustained for at least 10 days after stopping of antigen elicitation. Using NC/Nga mice, we investigated the contribution of scratching behavior to the development and maintenance of Dfb-induced dermatitis. In correlation with the increase in scratching behavior, erythema, hemorrhage, edema, scarring, erosion and excoriation were observed. Cutting off the hind toenails of mice exhibiting chronic skin lesions dramatically alleviated the dermatitis. From these findings, the onset of skin lesions and its chronically sustained course in Dfb-induced dermatitis in NC/Nga mice were closely associated with increased scratching behavior.

Regulation of the human PDZK1 expression by peroxisome proliferator-activated receptor alpha.

Although PDZK1 is a well-known adaptor protein, the mechanisms for its role in transcriptional regulation are largely unknown. The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. Previously, we established a tetracycline-regulated human cell line that can be induced to express PPARalpha and identified candidate target genes, one of which was PDZK1. In this study, we cloned and characterized the promoter region of the human pdzk1 gene and determined the PPAR response element. Finally, we demonstrate that endogenous PPARalpha regulates PDZK1 expression.

Antisense activity of 2',4'-BNA targeted to PPAR gamma in THP-1 and HCT116 cells.

2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) is a nucleic acid analogue that has high affinity binding to its complementary RNA. Peroxisome proliferator-activated receptor (PPAR) y belongs to the nuclear hormone receptor superfamily and is a drug target in the treatment of type 2 diabetes. Here, we show the antisense effects of 2',4'-BNA oligonucleotides against PPARy in the human monocytic leukaemia cell line THP-1 and the human colorectal tumor cell line HCT116.

[Application of two immunochromatographic test kits to screening of four kinds of allergic substances in imported foods].

We tested for four kinds of allergic substances (egg, milk, wheat and peanuts) in 52 imported processed foods using immunochromatographic test kits (ITK). ELISA was also employed to confirm the effectiveness of the ITK. Among 92 data from 23 samples, allergic substances were detected in 9 cases with one kind of ITK, but not with the ELISA test. Among 116 data from 29 samples, 6 were negative with one kind of ITK and but positive with the other ITK. These results suggested that these 4 kinds of allergic substances in imported foods can be detected by using a double-check method with two kinds of ITK.

Analysis of PPAR alpha function in human kidney cell line using siRNA.

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARalpha is mainly expressed in the liver, kidney, heart and muscle. PPARalpha activates fatty acid catabolism, stimulates gluconeogenesis and ketone body synthesis and is involved in the control of lipoprotein assembly. Although PPARalpha is well characterized in the liver, its physiological function is unknown in the kidney. To investigate the intimate function of PPARalpha in the kidney, we analyzed the target gene expression in human metastatic renal cell carcinoma cell line, Caki-1, using small interfering RNA (siRNA) against PPARalpha and real-time RT-PCR methods. We found that some selected genes (long-chain fatty-acid-CoA ligase (FACL1), carnitine palmitoyltransferase 1A (CPT1A), adipose differentiation-related protein (ADRP) and aquaporin 3 (AQP3)) were down-regulated by PPARalpha siRNA. These results suggest that PPARalpha regulates fatty acid metabolism and body water homeostasis in this cell line.

Immunization with 3-oxododecanoyl-L-homoserine lactone-protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection.

Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-L-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL-carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL-BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3 x 10(6) c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-alpha in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.

Synthesis of Pseudomonas quorum-sensing autoinducer analogs and structural entities required for induction of apoptosis in macrophages.

The synthesis of the analogs of N-3-oxododecanoyl-L-homoserine lactone (1) and their structure-activity relationship for the apoptotic induction in macrophages, P388D1 cells, are described. It was revealed that the position of the oxo group in the acyl side chain in addition to the presence of the L-homoserine lactone unit is crucial for the apoptosis-inducing activity. Furthermore, the long acyl side chains with hydrophobic distal ends are preferable for the activity.

Gene expression profiling of potential peroxisome proliferator-activated receptor (PPAR) target genes in human hepatoblastoma cell lines inducibly expressing different PPAR isoforms.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis. To identify human PPARs-responsive genes, we established tetracycline-regulated human hepatoblastoma cell lines that can be induced to express each human PPAR and investigated the gene expression profiles of these cells. RESULTS: The expression of each introduced PPAR gene was investigated using the various concentrations of doxycycline in the culture media. We found that the expression of each PPAR subtype was tightly controlled by the concentration of doxycycline in these established cell lines. DNA microarray analyses using these cell lines were performed with or without adding each subtype ligand and provided much important information on the PPAR target genes involved in lipid metabolism, transport, storage and other activities. Interestingly, it was noted that while ligand-activated PPARdelta induced target gene expression, unliganded PPARdelta repressed these genes. The real-time RT-PCR was used to verify the altered expression of selected genes by PPARs and we found that these genes were induced to express in the same pattern as detected in the microarray analyses. Furthermore, we analysed the 5'-flanking region of the human adipose differentiation-related protein (adrp) gene that responded to all subtypes of PPARs. From the detailed analyses by reporter assays, the EMSAs, and ChIP assays, we determined the functional PPRE of the human adrp gene. CONCLUSION: The results suggest that these cell lines are important tools used to identify the human PPARs-responsive genes.