Zoo animal manure as an overlooked reservoir of antibiotic resistance genes and multidrug-resistant bacteria.

Animal fecal samples collected in the summer and winter from 11 herbivorous animals, including sable antelope (SA), long-tailed goral (LTG), and common eland (CE), at a public zoo were examined for the presence of antibiotic resistance genes (ARGs). Seven antibiotics, including meropenem and azithromycin, were used to isolate culturable multidrug-resistant (MDR) strains. The manures from three animals (SA, LTG, and CE) contained 104-fold higher culturable MDR bacteria, including Chryseobacterium, Sphingobacterium, and Stenotrophomonas species, while fewer MDR bacteria were isolated from manure from water buffalo, rhinoceros, and elephant against all tested antibiotics. Three MDR bacteria-rich samples along with composite samples were further analyzed using nanopore-based technology. ARGs including lnu(C), tet(Q), and mef(A) were common and often associated with transposons in all tested samples, suggesting that transposons carrying ARGs may play an important role for the dissemination of ARGs in our tested animals. Although several copies of ARGs such as aph(3')-IIc, blaL1, blaIND-3, and tet(42) were found in the sequenced genomes of the nine MDR bacteria, the numbers and types of ARGs appeared to be less than expected in zoo animal manure, suggesting that MDR bacteria in the gut of the tested animals had intrinsic resistant phenotypes in the absence of ARGs.

Open-Label Placebo Treatment for Experimental Pain: A Randomized-Controlled Trial with Placebo Acupuncture and Placebo Pills.

Objective: An open-label placebo (OLP) is a placebo treatment in which the patient is aware that the treatment is a placebo. OLPs are considered effective for reducing pain, and previous studies have shown a stronger placebo effect for placebo acupuncture than for placebo pills. In this study, the authors compared the analgesic effects of OLP pills, OLP acupuncture, and a no treatment condition in healthy participants, and then examined the factors contributing to the OLP effect. Design: Randomized controlled crossover trial. Settings/Location: College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea. Subjects: 34 healthy participants. Intervention: Participants received three different treatments ("OLP-pill," "OLP-acupuncture," and "no treatment") on three separate days in random order. Outcome Measurements: Before and after the treatment, heat pain stimuli were applied to the participants' hands, and pain tolerance, intensity, and unpleasantness were measured using a visual analog scale (range, 0-10). Results: Data of 31 participants were included in the analysis. The authors found significant analgesic effects of the placebo pill and placebo acupuncture in the OLP condition. Regression analyses revealed that expectations regarding treatment and practitioner identity influenced the analgesic effects of OLP acupuncture. There was no adverse event. Conclusions: Expectations regarding treatment and practitioner identity influenced the analgesic effect of placebo acupuncture without deception. These findings provide new information regarding the cognitive factors underlying pharmacologic and nonpharmacologic treatments. Clinical Trial Registration Number: KCT0004928.

Killing effect of deinoxanthins on cyanobloom-forming Microcystis aeruginosa: Eco-friendly production and specific activity of deinoxanthins.

Cyanobacterial blooms caused mainly by Microcystis aeruginosa could be controlled using chemical and biological agents such as H2O2, antagonistic bacteria, and enzymes. Little is known about the possible toxic effects of bacterial membrane pigments on M. aeruginosa cells. Deinococcus metallilatus MA1002 cultured under light increased the production of several carotenoid-like compounds by upregulating two deinoxanthin biosynthesis genes: crtO and cruC. The deinoxanthin compounds were identified using thin-layer chromatography, high-performance liquid chromatography, and liquid chromatography-mass spectrometry. D. metallilatus was cultured with agricultural by-products under light to produce the deinoxanthin compounds. Soybean meal, from six tested agricultural by-products, was selected as the single factor for making an economical medium to produce deinoxanthin compounds. The growth of axenic M. aeruginosa PCC7806, as well as other xenic cyanobacteria such as Cyanobium gracile, Trichormus variabilis, and Dolichospermum circinale, were inhibited by the deinoxanthin compounds. Scanning electron microscopic images showed the complete collapse of M. aeruginosa cells under deinoxanthin treatment, probably due to its interference with cyanobacterial membrane synthesis during cellular elongation. Deinoxanthins appeared to be nontoxic to other non-cyanobacteria such as Acinetobacter, Pseudomonas, Methylobacterium, and Bacillus species, suggesting that it can be a novel candidate for preventing cyanobacterial blooms through its specific activity against cyanobacteria.

Raman spectroscopy reveals alteration of spore compositions under different nutritional conditions in Lysinibacillus boronitolerans YS11.

Little is known about final spores components when bacteria undergo sporulation under different nutrient conditions. Different degrees of resistance and germination rates were observed in the three types of spores of Lysinibacillus boronitolerans YS11 (SD, Spores formed in Difco sporulation medium™; SC and SF, Spores formed in an agricultural byproduct medium with 10 mM CaCl2 and with 10 mM FeSO4, respectively). Stronger UV resistance was recorded for SF with 1.8-2.3-fold greater survival than SC and SD under UV treatment. The three spore types showed similar heat resistances at 80°C, but survival rates of SC and SD were much higher (∼1,000 times) than those of SF at 90°C. However, germination capacity of SF was 20% higher than those of SD and SC on Luria-Bertani agar plates for 24 h. SF germinated more rapidly in a liquid medium with high NaCl concentrations than SC and SD, but became slower under alkaline conditions. Raman spectroscopy was used to analyze the heterogeneities in the three types of vegetative cells and their spores under different nutritional conditions. Exponentially grown-each vegetative cells had different overall Raman peak values. Raman peaks of SC, SD, and SF also showed differences in adenine and amide III compositions and nucleic acid contents. Our data along with Raman spectroscopy provided the evidence that spores formed under under different growth conditions possess very different cellular components, which affected their survival and germination rates.

Agricultural by-products and oyster shell as alternative nutrient sources for microbial sealing of early age cracks in mortar.

Bio-concrete using bacterially produced calcium carbonate can repair microcracks but is still relatively expensive due to the addition of bacteria, nutrients, and calcium sources. Agricultural by-products and oyster shells were used to produce economical bio-concrete. Sesame meal was the optimal agricultural by-product for low-cost spore production of the alkaliphilic Bacillus miscanthi strain AK13. Transcriptomic dataset was utilized to compare the gene expressions of AK13 strain under neutral and alkaline conditions, which suggested that NaCl and riboflavin could be chosen as growth-promoting factors at alkaline pH. The optimal levels of sesame meal, NaCl, and riboflavin were induced with the central composite design to create an economical medium, in which AK13 strain formed more spores with less price than in commercial sporulation medium. Calcium nitrate obtained from nitric acid treatment of oyster shell powder increased the initial compressive strength of cement mortar. Non-ureolytic calcium carbonate precipitation by AK13 using oyster shell-derived calcium ions was verified by energy-dispersive X-ray spectroscopy and X-ray diffraction analysis. Stereomicroscope and field emission scanning electron microscopy confirmed that oyster shell-derived calcium ions, along with soybean meal-solution, increased the bacterial survival and calcium carbonate precipitation inside mortar cracks. These data suggest the possibility of commercializing bacterial self-healing concrete with economical substitutes for culture medium, growth nutrient, and calcium sources.

Attentional Bias Toward Cupping Therapy Marks: An Eye-Tracking Study.

OBJECTIVE: Despite the many medical benefits, cupping therapy can be difficult for some patients due to unpleasant marks on the skin. As patients are afraid of the potential painful sensation from cupping therapy, the skin reactions might produce vigilance for treatment as pain-related information. We investigated whether individuals show negative emotions and attentional bias toward pain-related residual marks from cupping therapy on the body using an eye-tracking method. METHODS: Fifty pain-free volunteers were presented with four different kinds of visual stimulation, such as the back or face region and with or without cupping marks on the skin. A cupping and a control image were presented on one screen with one image on the left side of the screen and the other on the right (locations of the images were counterbalanced across participants). The eye movements of the participants were measured while they viewed the pictures. They completed the Empathy Quotient questionnaire before the experiment and evaluated the unpleasantness level to each image during the task. RESULTS: Images of the back and face with cupping marks were rated significantly more unpleasant and showed a significant attentional bias (significantly longer percentage fixation time) than the control images (attentional bias score: Back + cupping: 48.1 ± 2.8%; Back: -0.7 ± 3.4%; Face + cupping: 34.5 ± 2.5%; Face: -2.2 ± 2.9%). Individuals with greater empathy exhibited significantly higher unpleasantness (r = 0.323, p < 0.05) and less attentional bias (r = -0.279, p < 0.05) to the images with cupping marks. CONCLUSION: The skin reactions caused by cupping therapy evoked negative emotional responses as well as attentional bias to the reaction sites. Our findings suggest that the emotional and attentional responses to cupping therapy might reflect potential reluctance to this therapy.

Effects of Different Graphic Health Warning Types on the Intention to Quit Smoking.

Anti-smoking advertisements are widely used to demonstrate to smokers the harm of smoking, and graphic health warnings (GHWs) are expected to have a positive effect on the intention to quit smoking. This study investigated which type of GHW (health-related threat (H-GHW) vs. social threat (S-GHW)) is more effective. Two types of GHWs for tobacco were shown to 28 daily smokers and 25 non-smokers while measuring their eye movements using an eye tracker. The time spent fixating on the GHWs was measured as an index of attentional bias. Participants were also asked to evaluate the unpleasantness of the images. They stated their intention to quit smoking in response to each image in a separate session. Multiple regression analysis was used to identify the effects of psychosocial factors on the intention to quit smoking in smokers and the intention to remain as non-smokers in the non-smokers. Both smokers and non-smokers reported greater unpleasantness and cessation intentions in response to H-GHWs than to S-GHWs. Non-smokers found both types of GHWs more unpleasant than smokers did. No differences were found in gaze fixation on GHWs between the two groups. When smokers viewed S-GHWs, the intention to quit smoking was greater as they felt more unpleasant. For non-smokers, the intention to remain non-smokers was greater when they felt more unpleasant and when the attention to H-GHWs was lower. Different psychological factors in anti-smoking advertisements are involved in the intention to quit smoking in smokers and to maintain a non-smoking status in non-smokers. Different approaches should be used according to the types of warning (e.g., warnings emphasizing a negative influence on others or on their own health) in anti-smoking campaigns.

Cognitive and Emotional Aspects of Cupping Therapy.

Cupping therapy has recently gained public attention and is widely used in many regions. Some patients are resistant to being treated with cupping therapy, as visually unpleasant marks on the skin may elicit negative reactions. This study aimed to identify the cognitive and emotional components of cupping therapy. Twenty-five healthy volunteers were presented with emotionally evocative visual stimuli representing fear, disgust, happiness, neutral emotion, and cupping, along with control images. Participants evaluated the valence and arousal level of each stimulus. Before the experiment, they completed the Fear of Pain Questionnaire-III. In two-dimensional affective space, emotional arousal increases as hedonic valence ratings become increasingly pleasant or unpleasant. Cupping therapy images were more unpleasant and more arousing than the control images. Cluster analysis showed that the response to cupping therapy images had emotional characteristics similar to those for fear images. Individuals with a greater fear of pain rated cupping therapy images as more unpleasant and more arousing. Psychophysical analysis showed that individuals experienced unpleasant and aroused emotional states in response to the cupping therapy images. Our findings suggest that cupping therapy might be associated with unpleasant-defensive motivation and motivational activation. Determining the emotional components of cupping therapy would help clinicians and researchers to understand the intrinsic effects of cupping therapy.

Low-cost cultivation and sporulation of alkaliphilic Bacillus sp. strain AK13 for self-healing concrete.

The alkaliphilic, calcium carbonate precipitating Bacillus sp. strain AK13 can be utilized in concrete for self-repairing. A statistical experimental design was used to develop an economical medium for its mass cultivation and sporulation. Two types of screening experiment were first conducted to identify substrates that promote the growth of the AK13 strain: the first followed a one-factor-at-a-time factorial design and the second a two-level full factorial design. Based on these screening experiments, barley malt powder and mixed grain powder were identified as the substrates that most effectively promoted the growth of the AK13 strain from a range of 21 agricultural products and by-products. A quadratic statistical model was then constructed using a central composite design and the concentration of the two substrates was optimized. The estimated growth and sporulation of Bacillus sp. strain AK13 in the proposed medium were 3.08 ± 0.38 × 108 and 1.25 ± 0.12 × 108 CFU/ml, respectively, which meant that the proposed low-cost medium was approximately 45 times more effective than the commercial medium in terms of the number of cultivatable bacteria per unit price. The spores were then powdered via a spray-drying process to produce a spore powder with a spore count of 2.0 ± 0.7 × 109 CFU/g. The AK13 spore powder was mixed with cement paste, yeast extract, calcium lactate, and water. The yeast extract and calcium lactate generated the highest CFU/ml for AK13 at a 0.4:0.4 ratio compared to 0.4:0.25 (the original ratio of the B4 medium) and 0.4:0.8. Twenty-eight days after the spores were mixed into the mortar, the number of vegetative cells and spores of the AK13 strain had reached 106 CFU/g within the mortar. Cracks in the mortar under 0.29 mm were healed in 14 days. Calcium carbonate precipitation was observed on the crack surface. The mortar containing the spore powder was thus concluded to be effective in terms of healing micro-cracks.

Repeated Oral Administration of Human Serum Albumin Protects from the Cerebral Ischemia in Rat Brain Following MCAO.

Albumin is known to have neuroprotective effects. The protein has a long half-life circulation, and its effects can therefore persist for a long time to aid in the recovery of brain ischemia. In the present study, we investigated the neuroprotective effects of human serum albumin (HSA) on brain hemodynamics. Albumin is administrated using repeated oral gavage to the rodents. Sprague-Dawley rats underwent middle cerebral artery occlusion procedures and served as a stroke model. Afterwards, 25% human serum albumin (1.25 g/kg) or saline (5 ml/kg) was orally administrated for 2 weeks in alternating days. After 2 weeks, the rodents were assessed for levels of brain ischemia. Our testing battery consists of behavioral tests and in vivo optical imaging sessions. Modified neurological severity scores (mNSS) were obtained to assess the levels of ischemia and the effects of HSA oral administration. We found that the experimental group demonstrated larger hemodynamic responses following sensory stimulation than controls that were administered with saline. HSA administration resulted in more significant changes in cerebral blood volume following direct cortical electric stimulation. In addition, the mNSS of the treatment group was lower than the control group. In particular, brain tissue staining revealed that the infarct size was also much smaller with HSA administration. This study provides support for the efficacy of HSA, and that long-term oral administration of HSA may induce neuroprotective effects against brain ischemia.

Effect of a selective estrogen receptor modulator on bone formation in osteoporotic spine fusion using an ovariectomized rat model.

BACKGROUND CONTEXT: Selective estrogen receptor modulators (SERMs) are drugs used to treat osteoporosis. Selective estrogen receptor modulators improve bone mineral density and bone mechanical strength. However, there is a lack of data on the effect of SERMs on osteoporotic spinal fusion. PURPOSE: This study aimed to investigate whether treatment with a SERM enhances spinal fusion in an ovariectomized (OVX) rat model. STUDY DESIGN: An in vivo animal study was carried out. METHODS: Female Sprague Dawley rats (n=90) were OVX or sham-operated, and randomized into three groups: control (sham-operated+fusion procedure+saline administration), OVX (OVX+fusion procedure+saline administration), and SERM (OVX+fusion procedure+administration of SERM). Eight weeks after OVX, a bilateral lumbar spinal fusion procedure was performed using autologous iliac bone. In each group, gene expression was evaluated at 2, 4, and 8 weeks after the fusion procedure, histologic analysis was performed at 4 and 8 weeks after the procedure, and bone parameters were measured by micro-computed tomography at 2 days, 4 weeks, and 8 weeks after the procedure. RESULTS: Bone mineral density and trabecular quality of the vertebral body were significantly higher in the SERM group compared with the OVX group 16 weeks after OVX. The fusion rate and bone volume ratio were higher in the fusion bed of the SERM group compared with the OVX group 8 weeks after the fusion procedure. Real-time reverse transcription polymerase chain reaction at 4 and 8 weeks after the fusion procedure showed increased expression of the genes for osteoblast-related markers (alkaline phosphatase, osteocalcin, runt-related transcription factor 2, and transforming growth factor) in the SERM group compared with the OVX group. The OVX group showed sparse bone mass between transverse processes. By contrast, the SERM group exhibited a compact bridging fusion mass within the fusion bed at 8 weeks after the fusion procedure. CONCLUSIONS: Selective estrogen receptor modulator treatment improved the trabecular quality of the vertebral body, enhanced spinal fusion, and increased the amount of compact bone mass within the fusion bed in rats that had received an ovariectomy. Therapeutic SERM treatment may be recommended to improve the fusion rate in osteoporotic patients who undergo spinal fusion.

The real-time in vivo electrochemical measurement of nitric oxide and carbon monoxide release upon direct epidural electrical stimulation of the rat neocortex.

This study reports real-time, in vivo functional measurement of nitric oxide (NO) and carbon monoxide (CO), two gaseous mediators in controlling cerebral blood flow. A dual electrochemical NO/CO microsensor enables us to probe the complex relationship between NO and CO in regulating cerebrovascular tone. Utilizing this dual sensor, we monitor in vivo change of NO and CO simultaneously during direct epidural electrical stimulation of a living rat brain cortex. Both NO and CO respond quickly to meet physiological needs. The neural system instantaneously increases the released amounts of NO and CO to compensate the abrupt, yet transient hypoxia that results from epidural electrical stimulation. Intrinsic-signal optical imaging confirms that direct electrical stimulation elicits robust, dynamic changes in cerebral blood flow, which must accompany NO and CO signaling. The addition of l-arginine (a substrate for NO synthase, NOS) results in increased NO generation and decreased CO production compared to control stimulation. On the other hand, application of the NOS inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME), results in decreased NO release but increased CO production of greater magnitude. This observation suggests that the interaction between NO and CO release is likely not linear and yet, they are tightly linked vasodilators.

Study of the primo-vascular system and location-dependent oxygen levels for a mouse embryo.

The two major circulatory systems, the lymph system and the blood vessel system, play significant roles in controlling embryonic development. The primo-vascular system (PVS) was recently reported as an additional circulatory system in various animals. In this paper, the PVS in a mouse embryo was investigated. The structural characterization of the PVS in the mouse placenta and umbilical cord, which was visualized with the trypan blue staining technique, was focused on. The PVS was well_developed in the mouse placenta area. Using a nanopore-based amperometric oxygen sensor, the oxygen levels at four different areas of the embryonic brain, placenta, blood vessel, and primo-vessel of the PVS were measured. The relatively higher oxygen levels that were measured at the primo-vessels than at the brain and the placenta, while still lower than the oxygen level that was measured at the blood vessels, may suggest a role of PVS in oxygen transport.

Heterogeneity of skin surface oxygen level of wrist in relation to acupuncture point.

The distribution of partial oxygen pressure (pO(2)) is analyzed for the anterior aspect of the left wrist with an amperometric oxygen microsensor composed of a small planar Pt disk-sensing area (diameter = 25 µm). The pO(2) levels vary depending on the measurement location over the wrist skin, and they are systematically monitored in the analysis for both one-dimensional single line (along the wrist transverse crease) and two-dimensional square area of the wrist region. Relatively higher pO(2) values are observed at certain area in close proximity to the position of acupuncture points with statistical significance, indicating strong relationship between oxygen and acupuncture point. The used oxygen microsensor is sensitive enough to detect the pO(2) variation depending on the location. This study may provide information helpful to understand possible physiological roles of the acupuncture points.

Primo vascular system of murine melanoma and heterogeneity of tissue oxygenation of the melanoma.

Murine melanoma requires the complex development of lymphatic, vascular, and non-vascular structures. A possible relationship between the primo vascular system (PVS) and the melanoma metastasis has been proposed. In particular, the PVS may be involved in oxygen transport. Vasculogenic-like networks, similar to the PVS, have been found within melanoma tumors, but their functional relationship with the PVS and meridian structures are unclear. Herein, we report on the use of an electrochemical O(2) sensor to study oxygenation levels of melanoma tumors in mice. We consistently found higher tissue oxygenation in specific sites of tumors (n=5). These sites were strongly associated with vascular structures or the PVS. Furthermore, the PVS on the tumor surface was associated with adipose tissue. Our findings suggest that the PVS is involved in the regulation of metastasis.

Real-time in vivo simultaneous measurements of nitric oxide and oxygen using an amperometric dual microsensor.

This paper reports a real-time study of the codynamical changes in the release of endogenous nitric oxide (NO) and oxygen (O(2)) consumption in a rat neocortex in vivo upon electrical stimulation using an amperometric NO/O(2) dual microsensor. Electrical stimulation induced transient cerebral hypoxia due to the increased metabolic demands that were not met by the blood volume inside the stimulated cortical region. A NO/O(2) dual microsensor was successfully used to monitor the pair of real-time dynamic changes in the tissue NO and O(2) contents. At the onset of electrical stimulation, there was an immediate decrease in the cortical tissue O(2) followed by a subsequent increase in the cortical tissue NO content. The averages of the maximum normalized concentration changes induced by the stimulation were a 0.41 (±0.04)-fold decrease in the O(2) and a 3.6 (±0.9)-fold increase in the NO concentrations when compared with the corresponding normalized basal levels. The peak increase in NO was always preceded by the peak decrease in O(2) in all animals (n = 11). The delay between the maximum decrease in O(2) and the maximum increase in NO varied from 3.1 to 54.8 s. This rather wide variation in the temporal associations was presumably attributed to the sparse distribution of NOS-containing neurons and the individual animal's differences in brain vasculatures, which suggests that a sensor with fine spatial resolution is needed to measure the location-specific real-time NO and O(2) contents. In summary, the developed NO/O(2) dual microsensor is effective for measuring the NO and O(2) contents in vivo. This study provides direct support for the dynamic role of NO in regulating the cerebral hemodynamics, particularly related to the tissue oxygenation.