Microbiota transfer following liver surgery involves microbial extracellular vesicle migration that affects liver immunity.

BACKGROUND: Short-term perioperative administration of probiotics was shown to alleviate postoperative complications and promote liver recovery among patients undergoing resection for liver malignancy. The mechanisms by which probiotic bacteria effectively influence the gut microbiome composition during the perioperative time are controversial. Here, we aim to elucidate the short-term direct biological effect of probiotic microbiota-derived vesicles on host liver cells during the perioperative period. METHODS: Probiotic-derived vesicles (pbMVs) were administered postoperatively. pbMVs were isolated and characterized from probiotics, mainly from the bacteria genus Lactobacillus, Bifidobacterium, and Lactococcus. Mice underwent bile duct ligation, sham laparotomy (SHAM), or 70% partial hepatectomy (70%PH). pbMVs were tracked in vivo, and intrahepatic cellular and molecular aspects were analyzed by flow cytometry and qRT-PCR techniques. Liver sinusoidal endothelial cells (LSECs) analysis for Vascular Cell Adhesion Molecule-1(VCAM-1) expression following pbMV stimulation of cultured liver non-parenchymal cells which had been activated by LPS. RESULTS: The administered pbMV rapidly translocated to the liver after surgery. pbMV administrations following surgeries enhanced neutrophil clearance; there was a dramatic decline in the liver neutrophil-to-lymphocyte ratio Ly6G+/CD3+ and an increase in IL6 levels. pbMVs reduced intrahepatic VCAM1 and ICAM2 expression compared with control following SHAM and decrease in IL10 levels following 70%PH. The administration of pbMV improved liver regeneration 72 hours following surgical liver resection with a significant decrease in IL17 expression. pbMVs modulated VCAM-1 on liver sinusoidal endothelial cells in liver cell culture. CONCLUSIONS: Our study findings provide mechanistic insights into the liver-gut axis following surgery and illustrate how probiotic vesicles can reduce adhesion molecule expression and affect immune cell invasion and liver immunity, resulting in improved liver recovery following hepatic surgery.

High throughput optical modulation biosensing for highly sensitive and rapid detection of biomarkers.

Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many highly sensitive immunoassays, magnetic beads are used to capture fluorescently labeled target molecules. The target molecules are then quantified by detecting the fluorescent signal from individual beads, which is time consuming and requires a complicated and expensive detection system. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system, which uses a small permanent magnet to aggregate the beads into a small detection volume and eliminates background noise by steering a laser beam in and out of the cluster of beads. Shortening the aggregation, acquisition, and well-to-well scanning transition times enables reading a 96-well plate within 10 min. Using the ht-OMB system to detect human Interleukin-8, we demonstrated a limit of detection of 0.14 ng/L and a 4-log dynamic range. Testing 94 RNA extracts from 36 confirmed RT-qPCR SARS-CoV-2-positive patients (Ct≤40) and 58 confirmed RT-qPCR SARS-CoV-2-negative individuals resulted in 100% sensitivity and 100% specificity.

Optical modulation biosensing system for rapid detection of biological targets at low concentrations.

In many sensitive assays, target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Here, we introduce an optical modulation biosensing (OMB) system, which aggregates the beads into a small detection area and separates the signal from the background noise by manipulating the laser beam in and out of the cluster of beads. Using the OMB system to detect human interleukin-8, we demonstrated a limit of detection of 0.02 ng/L and a 4-log dynamic range. Using Zika-positive and healthy individuals' serum samples, we show that the OMB-based Zika IgG serological assay has 96% sensitivity and 100% specificity.

Configuration and Design of Electromagnets for Rapid and Precise Manipulation of Magnetic Beads in Biosensing Applications.

Rapid and precise manipulation of magnetic beads on the nano and micro scales is essential in many biosensing applications, such as separating target molecules from background molecules and detecting specific proteins and DNA sequences in plasma. Accurately moving magnetic beads back and forth requires at least two adjustable magnetic field gradients. Unlike permanent magnets, electromagnets are easy to design and can produce strong and adjustable magnetic field gradients without mechanical motion, making them desirable for use in robust and safe medical devices. However, using multiple magnetic field sources to manipulate magnetic beads presents several challenges, including overlapping magnetic fields, added bulk, increased cost, and reduced durability. Here, we provide a thorough analysis, including analytical calculations, numerical simulations, and experimental measurements, of using two electromagnets to manipulate magnetic beads inside a miniature glass cell. We analyze and experimentally demonstrate different aspects of the electromagnets' design, such as their mutual influence, the advantages and disadvantages of different pole tip geometries, and the correlation between the electromagnets' positions and the beads' aggregation during movement. Finally, we have devised a protocol to maximize the magnetic forces acting on magnetic beads in a two-electromagnet setup while minimizing the electromagnets' size. We used two such electromagnets in a small footprint magnetic modulation biosensing system and detected as little as 13 ng/L of recombinant Zika virus antibodies, which enables detection of Zika IgM antibodies as early as 5 days and as late as 180 days post symptoms onset, significantly extending the number of days that the antibodies are detectable.

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay.

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a "sandwich" complex. An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4-ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB-assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.

Improving the Sensitivity of Fluorescence-Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads.

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre- and post-photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high-sensitivity LX-200 system using photobleached magnetic beads, human interleukin-8 is detected with a threefold improvement in detection limit and signal-to-noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence-based assays.

Pulse Oximetry with Two Infrared Wavelengths without Calibration in Extracted Arterial Blood.

Oxygen saturation in arterial blood (SaO2) provides information about the performance of the respiratory system. Non-invasive measurement of SaO2 by commercial pulse oximeters (SpO2) make use of photoplethysmographic pulses in the red and infrared regions and utilizes the different spectra of light absorption by oxygenated and de-oxygenated hemoglobin. Because light scattering and optical path-lengths differ between the two wavelengths, commercial pulse oximeters require empirical calibration which is based on SaO2 measurement in extracted arterial blood. They are still prone to error, because the path-lengths difference between the two wavelengths varies among different subjects. We have developed modified pulse oximetry, which makes use of two nearby infrared wavelengths that have relatively similar scattering constants and path-lengths and does not require an invasive calibration step. In measurements performed on adults during breath holding, the two-infrared pulse oximeter and a commercial pulse oximeter showed similar changes in SpO2. The two pulse oximeters showed similar accuracy when compared to SaO2 measurement in extracted arterial blood (the gold standard) performed in intensive care units on newborns and children with an arterial line. Errors in SpO2 because of variability in path-lengths difference between the two wavelengths are expected to be smaller in the two-infrared pulse oximeter.

Detecting mutual awareness events.

It is quite common that multiple human observers attend to a single static interest point. This is known as a mutual awareness event (MAWE). A preferred way to monitor these situations is with a camera that captures the human observers while using existing face detection and head pose estimation algorithms. The current work studies the underlying geometric constraints of MAWEs and reformulates them in terms of image measurements. The constraints are then used in a method that 1) detects whether such an interest point does exist, 2) determines where it is located, 3) identifies who was attending to it, and 4) reports where and when each observer was while attending to it. The method is also applied on another interesting event when a single moving human observer fixates on a single static interest point. The method can deal with the general case of an uncalibrated camera in a general environment. This is in contrast to other work on similar problems that inherently assumes a known environment or a calibrated camera. The method was tested on about 75 images from various scenes and robustly detects MAWEs and estimates their related attributes. Most of the images were found by searching the Internet.

Temperature controlled burn generation system based on a CO2 laser and a silver halide fiber optic radiometer.

BACKGROUND AND OBJECTIVES: Experimental animal study of burns is dependent on a reliable burn generation system. Most of the experimental systems used today are unable to produce precise partial thickness burns. This limits the ability to study minor changes associated with burn care. The aim of the study was to develop a method for generating burns with a fixed depth using a CO2 laser burn generation system. MATERIALS AND METHODS: The burn generation system was composed of two components: a burn generation device and a temperature sensing and control system. These components were designed to operate together in order to keep a constant, predetermined skin surface temperature during prolonged burn generation. One hundred thirty-eight spot burns were generated on the back of five shaved 450 g male Wistar rats. The rat skin was exposed to a 70 degrees C for 5-60 seconds. The burned areas were excised and underwent evaluation by hematoxylin-eosin-stained slide microscopy. RESULTS: A linear correlation was found between the duration of exposure and the average burn depth (r = 0.93). This correlation is represented by the equation: burn depth in millimeters = 0.012x (duration in seconds of skin exposure at 70 degrees C). CONCLUSIONS: The fiber-optic-controlled laser burn generation system studied is a reliable tool for creating partial thickness as well as full thickness skin burns in rats.