Nano-optical method for transforming a single yeast cell using exogenous genes.

We report a highly efficient nano-optical method for transforming a single yeast cell using exogenous genes. It used laser tweezers or micromanipulators to immobilize the cell immersed in a DNA solution and created a transient nano-sized hole on its cell wall concurrently with laser scissors to deliver nano moles of DNA into the cell. With this method, one can directly transfer the naked DNA of exogenous genes into yeast cells for transformation. We successfully transformed S. cerevisiae yeasts respectively with GFP (Green Fluorescent Protein) plasmid and the nucleic acid extraction of a bacteria GF1 from the gut of Coptotermes formosanus termites. The experimental results demonstrated that the recombinants had high survival rate and transformation efficiency (28%). The recombinant GFP-yeast system showed green fluorescence for generations. GF1 DNA sequences were incorporated into the yeast genome as a heritable component with stable expression for multi-generations so that the recombinant GF1-yeast had a strong capability of digesting biomass as GF1. Our method would apply to different cells with cell walls for various gene transformations.

AI Aided Analysis on Saliva Crystallization of Pregnant Women for Accurate Estimation of Delivery Date and Fetal Status.

Saliva contains similar molecular components to serum. Analysis of saliva can provide important diagnostic information about the body. Here we report an artificial intelligence (AI) aided home-based method that can let pregnant women perform daily monitoring on their pregnant status and accurate prediction on their delivery date by the pattern analysis of their salivary crystals. The method was developed based on the information obtained from our investigation on the saliva samples of 170 pregnant women about the correlation of the salivary crystal pattern with pregnant age and fetal status. It demonstrated that the patterns of salivary crystallization could act as indicators of the pregnant age, fetal state, and some medical conditions of pregnant women. On this basis, with the aid of AI recognition and analysis of the fractal dimension and some characteristic crystals in the salivary crystallization, we performed estimation on the delivery date in both quantitative and qualitative manners. The accuracy of the prediction on 15 pregnant women was satisfactory: 100% delivering in the predicted week, 93.3% within the estimated three days, and 86.7% on the day as the prediction. We also developed a simple smartphone-based AI-aided salivary crystal imaging and analysis device as an auxiliary means to let pregnant women monitor their fetal status daily at home and predict their delivery date with adequate accuracy.

Effects of Low-Dose X-Ray on Cell Growth, Membrane Permeability, DNA Damage and Gene Transfer Efficiency.

BACKGROUND: We aimed to reveal if low dose X-rays would induce harmful or beneficial effect or dual response on biological cells and whether there are conditions the radiation can enhance gene transfer efficiency and promote cell growth but without damage to the cells. METHOD: A systematic study was performed on the effects of Kilo-V and Mega-V X-rays on the cell morphology, viability, membrane permeability, DNA damage, and gene transfection of 293 T and CHO cells. RESULTS: The Kilo-V X-rays of very low doses from 0.01 to 0.04 Gray in principle didn't induce any significant change in cell morphology, growth, membrane permeability, and cause DNA damage. The Mega-V X-ray had a damage threshold between 1.0 and 1.5 Gray. The 0.25 Gray Mega-V-X-ray could promote cell growth and gene transfer, while the 1.5 Gray Mega-V X-ray damaged cells. CONCLUSION: The very low dose of KV X-rays is safe to cells, while the effects of Mega-V-X-rays are dose-dependent. Mega-V-X-rays with a dose higher than the damage threshold would be harmful, that between 1.0 -1.5 Gray can evoke dual effects, whereas 0.25 Gray MV X-ray is beneficial for both cell growth and gene transfer, thus would be suitable for radiation-enhanced gene transfection.

A Smart Phone Based Handheld Wireless Spirometer with Functions and Precision Comparable to Laboratory Spirometers.

We report a smart phone based handheld wireless spirometer which uses a Lilly type sensing flowhead for respiratory signal acquisition and transmits the data to smartphone or other mobile terminals with Bluetooth signal transmission for data processing and result display. The developed spirometer was demonstrated to be able to detect flow rates ranging from 0-15 L/s with an accuracy of 4 mL/s, and can perform tests of flow volume (FV), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), peak expiratory flow (PEF), etc. By having the functions and precision comparable to laboratory spirometers, it satisfies the American Thoracic Society and European Respiratory Society (ATS/ERS) proposed performance requirements for spirometer. At the same time, it is low cost, light and handy, low power consumption battery-powered. The test of 12 cases of subjects using the developed spirometer also indicated that it was easy to use for both providers and patients, and suitable for the Point of Care Test (POCT) of chronic obstructive pulmonary disease (COPD) and asthma at general-practice settings and homes.

Technique of laser chromosome welding for chromosome repair and artificial chromosome creation.

Here we report a technique of laser chromosome welding that uses a violet pulse laser micro-beam for welding. The technique can integrate any size of a desired chromosome fragment into recipient chromosomes by combining with other techniques of laser chromosome manipulation such as chromosome cutting, moving, and stretching. We demonstrated that our method could perform chromosomal modifications with high precision, speed and ease of use in the absence of restriction enzymes, DNA ligases and DNA polymerases. Unlike the conventional methods such as de novo artificial chromosome synthesis, our method has no limitation on the size of the inserted chromosome fragment. The inserted DNA size can be precisely defined and the processed chromosome can retain its intrinsic structure and integrity. Therefore, our technique provides a high quality alternative approach to directed genetic recombination, and can be used for chromosomal repair, removal of defects and artificial chromosome creation. The technique may also have applicability on the manipulation and extension of large pieces of synthetic DNA.

Versatile on-stage microfluidic system for long term cell culture, micromanipulation and time lapse assays.

We report here a versatile on-stage microfluidic cell culture and assay system which is compatible with different microscopes and sensors, can simultaneously perform steps of long term cell culture, high throughput time lapse cell assays/imaging, and cell micromanipulations. With the system, we cultured a variety of cells for different periods of time and monitored their cell morphology, migration and division. We also performed a series non-invasive real time in situ time lapse assays and micromanipulations on different cells. They include: the first time lapse imaging and measurements on the instantaneous variations of morphology, biomechanical properties and the intracellular protein of human red blood cells in responding to pH fluctuation, drug action and electromagnetic radiation; the first continuous time lapse Raman micro-spectroscopy on a CHO cell in different phases of its entire life cycles; the micro-transfection of GFP into B16 cells and the follow up observation of the cell's morphology and expressed GFP fluorescence varying with incubation time and cell generations. The performance of these experiments not only demonstrated the capability of the system, but also proposed a variety of novel methods for obtaining time- and spatially-resolved information about the cellular and molecular heterogeneity and transformation during development or stimulations.

Easy and rapid multi-pass detection of antigen and antibody with micro-lens sensors.

We introduce a micro-lens imaging method that can perform easy and rapid multi-pass detection of antigen/antibody (Ag/Ab) without the requirement of any labeling, expensive enzymes, pre-immobilization/modification, and post-washing. Our method detects Ag or Ab presenting in solutions in a quantitative or qualitative manner by using micro-lens as the sensor to monitor the refractive index variation of the solutions during the primary stage of Ag-Ab reaction. The detection can be taken rapidly and finished in two minutes, while requires very low sample volume (several micron liters) and its detection limit can be as low as ∼pg/mL. The method is also able to provide kinetic and thermodynamic parameters of Ag-Ab reactions. The detections of ten Ag-Ab systems and two kinds of clinical samples demonstrated that our method is of high sensitivity, accuracy, reliability and permitting on-site analysis.

Simultaneous Determination of Glass Transition Temperatures of Several Polymers.

AIMS: A simple and easy optical method is proposed for the determination of glass transition temperature (Tg) of polymers. METHODS & RESULTS: Tg was determined using the technique of microsphere imaging to monitor the variation of the refractive index of polymer microsphere as a function of temperature. It was demonstrated that the method can eliminate most thermal lag and has sensitivity about six fold higher than the conventional method in Tg determination. So the determined Tg is more accurate and varies less with cooling/heating rate than that obtained by conventional methods. The most attractive character of the method is that it can simultaneously determine the Tg of several polymers in a single experiment, so it can greatly save experimental time and heating energy. CONCLUSION: The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes. Therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.

Restoring the youth of aged red blood cells and extending their lifespan in circulation by remodelling membrane sialic acid.

Membrane sialic acid (SA) plays an important role in the survival of red blood cells (RBCs), the age-related reduction in SA content negatively impacts both the structure and function of these cells. We have therefore suggested that remodelling the SA in the membrane of aged cells would help recover cellular functions characteristic of young RBCs. We developed an effective method for the re-sialylation of aged RBCs by which the cells were incubated with SA in the presence of cytidine triphosphate (CTP) and α-2,3-sialytransferase. We found that RBCs could be re-sialylated if they had available SA-binding groups and after the re-sialylation, aged RBCs could restore their membrane SA to the level in young RBCs. Once the membrane SA was restored, the aged RBCs showed recovery of their biophysical and biochemical properties to similar levels as in young RBCs. Their life span in circulation was also extended to twofold. Our findings indicate that remodelling membrane SA not only helps restore the youth of aged RBCs, but also helps recover injured RBCs.

How cell number and cellular properties of blood-banked red blood cells of different cell ages decline during storage.

AIMS: Numerous studies have suggested that transfusion of red blood cells (RBCs) stored over a long period of time may induce harmful effects due to storage-induced lesions. However, the underlying mechanisms responsible for this damage have not been identified. Furthermore, it is unclear why and how up to 30% of long-stored RBCs disappear from the circulation within 24 hours after transfusion. The aim of this study was to determine how the cell number of RBCs of different ages changes during storage and how these cells undergo cumulative structural and functional changes with storage time. METHODS AND RESULTS: We used Percoll centrifugation to fractionate the RBCs in blood bank stored RBC units into different aged sub-populations and then measured the number of intact cells in each sub-population as well the cells' biomechanical and biochemical parameters as functions of the storage period. We found that the RBC units stored for ≤ 14 days could be separated into four fractions: the top or young cell fraction, two middle fractions, and the lower or old fraction. However, after 14 days of storage, the cell number and cellular properties declined rapidly whereby the units stored for 21 days only exhibited the three lower fractions and not the young fraction. The cell number within a unit stored for 21 days decreased by 23% compared to a fresh unit and the cells that were lost had hemolyzed into harmful membrane fragments, microparticles, and free hemoglobin. All remaining cells exhibited cellular properties similar to those of senescent cells. CONCLUSION: In RBC units stored for greater than 14 days, there were fewer intact cells with no healthy cells present, as well as harmful membrane fragments, microparticles, and free hemoglobin. Therefore, transfusion of these stored units would not likely help patients and may induce a series of clinical problems.

[Surface-enhanced Raman spectra analysis of trace degradation products from goat horn].

Nano-silver colloid was synthesized by using microwave method on the mixtures of sodium citrate solution and silver nitrate solution. The method has advantages of fast heating speed, uniform temperature distribution and easily controlled reaction conditions. The sizes and size distributions of the silver particles were characterized by means of quasi-elastic laser scattering (QLS). The average particles size was (53.27 +/- 2.65) nm and the size of the particles was mainly distributed around 56 nm. Surface-enhanced Raman spectra of the degradation products from goat horn were obtained with silver colloid as active substrate. It was observed that the Raman signal of SERS was enhanced significantly compared with that of regular Raman spectrum, especially at the Raman bands of 659, 830, 850, 929, 999, 1 028, 1 280, 1 439 and 1 599 cm(-1) which reflect the biochemical components in degradation products. The characteristic Raman bands of degradation products from goat horn were preliminary assigned. The assignments showed that the main constituents of the degradation products from goat horn were amino acids and polypeptides. It was for the first time that Surface-enhanced Raman spectroscopy was used to detect trace degradation products from the horns. Raman signal enhancement can be obtained with high sensitivity for the trace concentrations as low as ppm level. It is concluded that surface-enhanced Raman spectroscopy can provide a fast, direct and precise detecting method for the detection of trace degradation solution from horns.

KrF excimer laser precision machining of hard and brittle ceramic biomaterials.

KrF excimer laser precision machining of porous hard-brittle ceramic biomaterials was studied to find a suitable way of machining the materials into various desired shapes and sizes without distorting their intrinsic structure and porosity. Calcium phosphate glass ceramics (CPGs) and hydroxyapatite (HA) were chosen for the study. It was found that KrF excimer laser can cut both CPGs and HA with high efficiency and precision. The ablation rates of CPGs and HA are respectively 0.081 µm/(pulse J cm(-2)) and 0.048 µm/(pulse J cm(-2)), while their threshold fluences are individually 0.72 and 1.5 J cm(-2). The cutting quality (smoothness of the cut surface) is a function of laser repetition rate and cutting speed. The higher the repetition rate and lower the cutting speed, the better the cutting quality. A comparison between the cross sections of CPGs and HA cut using the excimer laser and using a conventional diamond cutting blade indicates that those cut by the excimer laser could retain their intrinsic porosity and geometry without distortion. In contrast, those cut by conventional machining had distorted geometry and most of their surface porosities were lost. Therefore, when cutting hard-brittle ceramic biomaterials to prepare scaffold and implant or when sectioning them for porosity evaluation, it is better to choose KrF excimer laser machining.

Pathway and mechanism of pH dependent human hemoglobin tetramer-dimer-monomer dissociations.

Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer → dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer → monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or re-association reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development.

Microstructure and mechanical properties of horns derived from three domestic bovines.

The microstructure and mechanical properties of horns derived from three domestic bovines (buffalo, cattle and sheep) were examined. The effects of water content, sampling position and orientation of three bovid horns on mechanical properties were systematically investigated by uniaxial tension and micron indentation tests. Meanwhile, the material composition and metal element contents were determined by Raman spectroscopy and elemental analysis respectively, and the microstructures of the horns were measured by scanning electron microscopy (SEM). Results show that the mechanical properties of horns have negative correlation with water contents and depend on sampling position and orientation. The spatial variations of the mechanical properties in horns are attributed to the different keratinization degrees in the proximal, middle and distal parts. And the mechanical properties of horns in the longitudinal direction are better than those in transverse. Among the three kinds of horns, the mechanical properties of buffalo horn are the best, followed by cattle horn, and those in sheep horn are the worst. This is due to the differences in material composition, metal element, and the microstructures of the horns. But the mechanical properties of buffalo horns are not dependent on the source of the buffalo. Therefore, regular engineered buffalo keratinous materials with standard mechanical properties can be obtained from different buffalo horns by using proper preparation methods.

Characteristic variation of α-fetoprotein DNA nanometer-range irradiated by iodine-125.

To obtain the characteristic variation of structure and functional groups of α-fetoprotein (AFP) DNA irradiated by iodine-125((125)I), the AFP antisense oligonucleotide labeled with various radioactivity dose (125)I was mixed with the AFP DNA in a simulated polymerase chain reaction temperature condition. After the mixtures were irradiated by the (125)I from 2 to 72 hours, the mutation of the biogenic conformation and functional groups of the irradiated DNA were investigated using laser Raman spectroscopy. The shifted peak and the decreased intensity of the characteristic Raman spectra were found, which demonstrated that the structure of the phosphodiester linkage was broke, the pyridine and purine bases in DNA emerged and damaged. The model of gene conformation changed from form B to form C spectrum after the nanometer-range irradiation with (125)I from 2 to 24 hours. The damage of local pyridine and purine bases gradually increased along with the accumulation of irradiation, and the bases and ribosome were finally dissociated and stacked.

Micro optical power meter for direct in situ measurement of light transmitted from microscopic systems and focused on micro-samples.

This paper reports a micro optical power meter which is able to perform effective and precise measurement on the optical power at the focus of different microscopic systems. The power meter can be easily placed on the stages of different microscopes and even partly immersed into solution to directly measure the optical power transmitted from the microscope objective and focused on the sample suspended in solution. The testing experiments demonstrated that the power meter has the characteristics of high precision, excellent linearity, high sensitivity, good stability, and high responding speed. It can accurately measure power levels from 0.1 to 50 mW in visible wavelength in various conditions and environments, which may encounter in practical applications. The optical power measurements using the power meter performed in some biological cell culturing solutions and in air for the same laser light reveal the first time that the powers measured in solutions were about 5%-8% greater than that measured in air at the same position. This not only suggests the necessity of performing direct measurement in situ in solution to obtain the real optical power projected on the suspended samples, but also indicates that such a micro optical power meter can meet almost all the requirements of optical power measurement in different fields from biomedicine to material sciences.

Reactive oxygen species formation and apoptosis in human peripheral blood mononuclear cell induced by 900 MHz mobile phone radiation.

We demonstrate that reactive oxygen species (ROS) plays an important role in the process of apoptosis in human peripheral blood mononuclear cell (PBMC) which is induced by the radiation of 900 MHz radiofrequency electromagnetic field (RFEMF) at a specific absorption rate (SAR) of ~0.4 W/kg when the exposure lasts longer than two hours. The apoptosis is induced through the mitochondrial pathway and mediated by activating ROS and caspase-3, and decreasing the mitochondrial potential. The activation of ROS is triggered by the conformation disturbance of lipids, protein, and DNA induced by the exposure of GSM RFEMF. Although human PBMC was found to have a self-protection mechanism of releasing carotenoid in response to oxidative stress to lessen the further increase of ROS, the imbalance between the antioxidant defenses and ROS formation still results in an increase of cell death with the exposure time and can cause about 37% human PBMC death in eight hours.

Easy method to determine refractive indices of microspheres and in micro-regions of inhomogeneous media.

We describe an innovative method which can accurately determine the refractive index (RI) of individual microspheres by immersing the microspheres in a medium and analyzing their phase-contrast microscopic images. Compared with the current techniques for microsphere RI measurement, the method has several advantages: it is simple and easy and it cannot only measure the RI of each individual microsphere but also perform measurement simultaneously on all the microspheres in the same field of view. In measurement, microspheres are not required to be suspended in a specific liquid but in any medium with known RI which is appropriate for the microspheres or even just in atmosphere. By using microspheres with known RI as sensors, the method can also be used for rapid in situ measurement of the local RI of inhomogeneous media. In this paper, we describe the principle of the method and the experiments of using the method to measure the RI of individual microspheres. Its applications for sensing instantaneous RI/concentration/temperature variation in critical situations such as anywhere in mixing flows or living biological specimens are also presented.

[Study of spectrum processing method for Raman microscopy on single living cell].

To study the spectrum processing methods for Raman microscopy on single living cell and develop the pre-process techniques for Raman spectrum of single living cell to enhance the signal to noise ratio, sensitivity, and decrease the fluorescence influence, wiping off the cosmic rays was used to improve the spectrum. The spectra classification, spectra average and filtration were applied to enhance signal to noise ratio. The fluorescence was depressed for quantity analysis or utilized for analysis by comparing the background and the spectra. Results show that (1) comparing the spectra with short exposure time and more scans can wipe off the cosmic rays effectively. (2) the spectra classification, spectra average and filtration can improve the quality of spectra and can show some weak and sensitive bands. (3) sometimes the fluorescence has useful information. It is concluded that the proposed techniques for Raman spectrum of single cell in this paper can show the sensitive and weak intensity peaks and reflect the information of molecules structures very well.

Human red blood cell aging: correlative changes in surface charge and cell properties.

Red blood cells (RBCs) during microcirculation, aging and storage, lose N-acetylneuraminic acid (NANA) and other biomaterials thereby altering cell structures, some properties and functions. Such cell damage very likely underlies the serious adverse effects of blood transfusion. However, a controversy has remained since 1961-1977 as to whether with aging, the RBCs, suffering loss of NANA, do have a decreased charge density. Any correlation between the changes in the cell properties with cell aging is also not clear. Therefore, to remove the ambiguity and uncertainty, we carried out multiparameteric studies on Percoll fractions of blood of 38 volunteers (lightest-young-Y-RBCs, densest-old-O-RBCs, two middle fractions).We found that there were striking differences between the properties of Y-RBCs and O-RBCs. The ζ-potential of Y-RBCs decreased gradually with aging. Studies in parallel on RBC fractions incubated with both positively charged quantum dots and Sambucus Nigra-fluorescein isothiocyanate (FITC) along with their ζ-potentials provide for the first time direct visual evidence about the lesser amount of charge density and NANA on O-RBCs, and a collinear decrease in their respective ζ-potentials. Close correlation was found between the surface charge on an aging RBC and its structure and functions, from the cell morphology, the membrane deformability to the intracellular Hb structure and oxidation ability. This quantitative approach not only clarifies the picture but also has implications in biology and medicine.