Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Optimization of nitrogen source for Bifidobacterium bifidum using response surface methodology

In order to improve the viable counts of Bifidobacterium bifidum BB01 in the liquid medium, the Central Composite Design (CCD) was used to optimize the nitrogen source in the medium of B. bifidum BB01. The results showed that the nitrogen source composition of B. bifidum BB01 was: peptone 0.9%, yeast extracts 0.3%, beef paste 0.7%. Under the optimal conditions, the viable counts of B. bifidum BB01 reached (2.49±0.06)×109CFU/mL after cultured at 18h, which was 42.97% higher than MRS (lactose), and 12.85% higher than the optimized MRS medium (carbon source and prebiotics were optimized). Therefore, the CCD used in this study is workable for promoting the growth of B. bifidum BB01

Application of central composite design to optimize the amount of carbon source and prebiotics for Bifidobacterium bifidum BB01

The objective of the present study was to obtain the optimum proportion of the carbon source and prebiotics for Bifidobacterium bifidum BB01 by the central composite design (CCD). The effect of carbon source (lactose) and two prebiotics (inulin and fructooligosaccharides) on the BB01 were observed by measuring the OD600 value, pH value and the viable counts at 18h. The final optimized concentrations of carbon source and prebiotics were: lactose 1.6%, inulin 0.26%, and fructooligosaccharides 0.22%. The result indicates that the growth of B. bifidum BB01 shows an significant increase in the optimized culture medium (p < 0.05), the OD600 value reached 1.434 at 18h, which increased 6.58% compared to the control. And the viable counts of B. bifidum BB01 increased 24.36% and reached (2.17±0.06) ×109cfu/mL. The results show that the optimization of the carbon source and prebiotics using CCD in this study is workable and necessary

Reactive oxygen species inhibits Listeria monocytogenes invasion into HepG2 epithelial cells

Listeria monocytogenes (Lm) can colonize human gastrointestinal tract and subsequently cross the intestinal barrier. Reactive oxygen species (ROS) are produced by NADPH oxidase. However, the role of ROS in bacterial invasion remains to be less understood. Herein, we investigated the impact of ROS on Lm invasion to HepG2 using NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), as well as the ROS scavenger, N‐acetyl cysteine (NAC). Our results showed that inhibiting ROS increased the invasive capability of Lm. Moreover, after Lm infection, inflammatory cytokines such as tumor necrosis factor alpha (TNF‐α) and interleukin 1beta (IL‐1β) in HepG2 were significantly upregulated. However, after inhibiting ROS, the expression levels of TNF‐α and IL‐1β were downregulated, indicating a failure of host cells to activate the immune mechanism. Taken together, ROS in Lm might be as a signal for host cells to sense Lm invasion and then stimulate cells to activate the immune mechanism

The Effect of Feeding Neutralizer on the Growth of Bifidobacterium Bifidum

In order to investigative the effect of different neutralizers and their feeding time on culture of Bifidobacterium bifidum BB01, and pH, OD and viable count of B. bifidumBB01 in the medium in different time were measured. The results indicated that the NaOH solution was the optimum neutralizer compared with the others, and feeding time of the neutralizer to B. bifidum BB01 was 13 hours after inoculation. Furthermore, the OD value and viable count reached maximum at 21h (OD value= 1.667) and 20h (viable count: (3.52±0.046) ×109 CFU/mL) after the NaOH solution was added to the medium, respectively. In addition, the maximum OD value implied that the logarithmic phase of B. bifidum BB01 was delayed compared with the control and the viable count were 29.26% larger than the control group. The result of the study provides a method and an important basis for improving the viable counts of B. bifidum BB01

Homocysteine exaggerates microglia activation and neuroinflammation through microglia localized STAT3 overactivation following ischemic stroke

Abstract Background Elevated plasma homocysteine (Hcy) levels have been indicated as a strong and modifiable risk factor of ischemic stroke; the previous studies have shown that exposure to Hcy activates cultured microglia. However, whether neurotoxicity of Hcy involves microglia activation following brain ischemia and the underlying mechanisms remains incompletely understood. Methods The cerebral damage was evaluated by staining with 2,3,5-triphenyltetrazolium chloride, hematoxylin-eosin, and Fluoro Jade B. The activation state of microglia was assessed via immunoreaction using the microglial markers Iba1 and OX-42. Then, the inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were examined by Western blot analysis and fluorescence immunohistochemistry. Results Elevated Hcy level augmented brain damage and neural cell toxicity in the brain cortex and the dentate gyrus region of the hippocampus after cerebral ischemia/reperfusion. Meanwhile, Hcy activated microglia and induced the expression of the inflammatory factors such as TNF-α and IL-6. Moreover, Hcy caused an increase in pSTAT3 expression which occurs in microglial cells. AG490, a JAK2-STAT3 inhibitor, effectively inhibited the phosphorylation of STAT3, microglial cell activation and the secretion of IL-6, TNF-α raised by Hcy treatment. Conclusions STAT3 signaling pathway located in microglia plays a critical role in mediating Hcy-induced activation of microglia and neuroinflammation in rat MCAO model. This suggests the feasibility of targeting the JAK2/STAT3 pathway as an effective therapeutic strategy to alleviate the progression of Hcy-associated ischemia stroke

Natural Dietary Compound Xanthohumol Regulates the Gut Microbiota and Its Metabolic Profile in a Mouse Model of Alzheimer’s Disease

Discovering new and effective drugs for the treatment of Alzheimer’s disease (AD) is a major clinical challenge. This study focuses on chemical modulation of the gut microbiome in an established murine AD model. We used the 16S rDNA sequencing technique to investigate the effect of xanthohumol (Xn) on the diversity of intestinal microflora in 2-month- and 6-month-old APP/PS1 mice, respectively. APP/PS1 and wild-type mice were treated by gavage with corn oil with or without Xn every other day for 90 days. Prior to and following treatment, animals were tested for spatial learning, cognitive and memory function. We found Xn reduced cognitive dysfunction in APP/PS1 mice and significantly regulated the composition and abundance of gut microbiota both in prevention experiments (with younger mice) and therapeutic experiments (with older mice). Differential microflora Gammaproteobacteria were significantly enriched in APP/PS1 mice treated with Xn. Nodosilineaceae and Rikenellaceae may be the specific microflora modulated by Xn. The penicillin and cephalosporin biosynthesis pathway and the atrazine degradation pathway may be the principal modulation pathways. Taken together, oral treatment with Xn may have a neuroprotective role by regulating the composition of intestinal microflora, a result that contributes to the scientific basis for a novel prophylactic and therapeutic approach to AD

Low‐voltage DC building distribution and utilization system and its implementation in China southern grid

Abstract The demand‐side DC electricity‐using equipment and newly integrated renewables are driving the transformation of power distribution and utilization mode. The building system based on DC technology is emerging as a promising option. In the low‐voltage DC building distribution and utilization system (LVDCBDUS), global energy optimization management and operational control arrangement are key components. To obtain exemplary achievements of those, two different DC building energy management system (DC BEMS) integration schemes are investigated according to the respective features and application‐required functions of various system networking structures. Centralized and decentralized control strategies are presented and discussed for buildings with AC–DC transformation and newly built LVDCBDUSs. On this basis, the centralized DC BEMS and operational control strategy are applied to the first multi‐scenario low‐carbon city‐based future building project—Shenzhen IBR Future Complex. The operation data are recorded and analysed. Problems encountered during the implementation are summarized, and requirements of converter equipment, new technologies and marketization are further discussed to promote the high‐quality development of the LVDCBDUS

Fine Structure of Tibetan Kefir Grains and Their Yeast Distribution, Diversity, and Shift

<div><p>Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and <i>in situ</i> hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – <i>Saccharomyces cerevisiae</i>, <i>Kluyveromyces marxianus</i>, and <i>Yarrowia lipolytica</i>; (iii) <i>S. cerevisiae</i> is the stable predominant yeast species, while the composition of <i>Kluyveromyces</i> and <i>Yarrowia</i> are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between <i>S. cerevisiae</i> and <i>Lactobacillus</i> bacteria in TKGs.</p></div

Phylotypes of yeast community in TKGs.

<p>N-1: NCTKG-1, N-2: NCTKG-2, A-1: ACTKG-1, A-2: ACTKG-2. The NCTKG-2 and ACTKG-2 are the samples cultivated for 10 months more than the NCTKG-1 and ACTKG-1. A sequence identify of ≥99% is considered the identification to the species level; that of ≥97% to the genus level <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101387#pone.0101387-Kurtzman2" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101387#pone.0101387-Garner1" target="_blank">[41]</a>.</p