Achromatization method for multichannel fluorescence imaging systems

An achromatization method optimized for dual-channel imaging is developed. Dichroic mirrors are employed to split and recombine narrowband signals, and separation between catoptric components is used to minimize the longitudinal chromatic shift. An achromatic system based on this principle could be built from singlet lenses, since refractive element properties such as dispersion and power are not utilized to optimize wavelength-dependent performance. To demonstrate the validity of the proposed solution, a prototype miniature fluorescence microscope optimized for two emission lines of acridine orange (525 and 650 nm) is built. To reduce the cost and accelerate assembly, the system is built from commercially available optical components. The optical train consisted of two plastic singlet lenses combined with a pair of dichroic mirrors. Optical performance of the prototype is evaluated by imaging a bar line target at both design wavelengths. To demonstrate the potential of the proposed design strategy, the achromatic system prototype is used to measure a two-part white blood cells differential count on a venous blood sample. Data from the prototype fluorescence microscope are compared against results from a commercially available blood analyzer, and the difference between both instruments is within 20%

Ti6Ta4Sn alloy and subsequent scaffolding for bone tissue engineering

Porous titanium (Ti) and titanium alloys are promising scaffold biomaterials for bone tissue engineering, because they have the potential to provide new bone tissue ingrowth abilities and low elastic modulus to match that ofnatural bone. In the present study, a new highly porous Ti6Ta4Sn alloy scaffold with the addition of biocompatible alloying elements (tantalum (Ta) and tin (Sn)) was prepared using a space-holder sintering method. Thestrength of the Ti6Ta4Sn scaffold with a porosity of 75% was found to be significantly higher than that of a pure Ti scaffold with the same porosity. The elastic modulus of the porous alloy can be customized to match that ofhuman bone by adjusting its porosity. In addition, the porous Ti6Ta4Sn alloy exhibited an interconnected porous structure, which enabled the ingrowth of new bone tissues. Cell culture results revealed that human SaOS2osteoblast-like cells grew and spread well on the surfaces of the solid alloy, and throughout the porous scaffold. The surface roughness of the alloy showed a significant effect on the cell behavior, and the optimum surfaceroughness range for the adhesion of the SaOS2 cell on the alloy was 0.15 to 0.35 mm. The present study illustrated the feasibility of using the porous Ti6Ta4Sn alloy scaffold as an orthopedic implant material with a specialemphasis on its excellent biomechanical properties and in vitro biocompatibility with a high preference by osteoblast-like cells.<br /

Differentiating between live and deadﾠMycobacterium smegmatisﾠusing autofluorescence

While there have been research efforts to find faster and more efficient diagnostic techniques for tuberculosis (TB), it is equally important to monitor a patient's response to treatment over time, especially with the increasing prevalence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) TB. Between sputum smear microscopy, culture, and GeneXpert, only culture can verify viability of mycobacteria. However, it may take up to six weeks to grow Mycobacterium tuberculosis (Mtb), during which time the patient may have responded to treatment or the mycobacteria are still viable because the patient has MDR or XDR TB. In both situations, treatment incurs increased patient costs and makes them more susceptible to host-drug effects such as liver damage. Coenzyme Factor 420 (F420) is a fluorescent coenzyme found naturally in mycobacteria, with an excitation peak around 420 nm and an emission peak around 470 nm. Using Mycobacterium smegmatis, we show that live and dead mycobacteria undergo different rates of photobleaching over a period of 2 min. These preliminary experiments suggest that the different photobleaching rates could be used to help monitor a patient's response to TB treatment. In future studies, we propose to describe these experiments with Mtb as both M. smegmatis and Mtb use F420

Sleep-wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here, we demonstrated that PR-domain containing protein 13 (Prdm13)+ neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not in old mice. Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control. Furthermore, DMH-specifi

Medical textiles as vascular implants and their success to mimic natural arteries

Vascular implants belong to a specialised class of medical textiles. The basic purpose of a vascular implant (graft and stent) is to act as an artificial conduit or substitute for a diseased artery. However, the long-term healing function depends on its ability to mimic the mechanical and biological behaviour of the artery. This requires a thorough understanding of the structure and function of an artery, which can then be translated into a synthetic structure based on the capabilities of the manufacturing method utilised. Common textile manufacturing techniques, such as weaving, knitting, braiding, and electrospinning, are frequently used to design vascular implants for research and commercial purposes for the past decades. However, the ability to match attributes of a vascular substitute to those of a native artery still remains a challenge. The synthetic implants have been found to cause disturbance in biological, biomechanical, and hemodynamic parameters at the implant site, which has been widely attributed to their structural design. In this work, we reviewed the design aspect of textile vascular implants and compared them to the structure of a natural artery as a basis for assessing the level of success as an implant. The outcome of this work is expected to encourage future design strategies for developing improved long lasting vascular implants

Achromatization method for multichannel fluorescence imaging systems

An achromatization method optimized for dual-channel imaging is developed. Dichroic mirrors are employed to split and recombine narrowband signals, and separation between catoptric components is used to minimize the longitudinal chromatic shift. An achromatic system based on this principle could be built from singlet lenses, since refractive element properties such as dispersion and power are not utilized to optimize wavelength-dependent performance. To demonstrate the validity of the proposed solution, a prototype miniature fluorescence microscope optimized for two emission lines of acridine orange (525 and 650 nm) is built. To reduce the cost and accelerate assembly, the system is built from commercially available optical components. The optical train consisted of two plastic singlet lenses combined with a pair of dichroic mirrors. Optical performance of the prototype is evaluated by imaging a bar line target at both design wavelengths. To demonstrate the potential of the proposed design strategy, the achromatic system prototype is used to measure a two-part white blood cells differential count on a venous blood sample. Data from the prototype fluorescence microscope are compared against results from a commercially available blood analyzer, and the difference between both instruments is within 20%

Parental beliefs, infant temperament, and marital quality: Associations with infant–mother and infant–father attachment.

The present research examined parental beliefs about the importance of the paternal caregiving role, mothers’ and fathers’ reports of infant temperament, and observed marital quality as predictors of infant-mother and infant-father attachment security, over and above the effects of parental sensitivity. Infants’ attachment security to mothers and fathers were observed in the Strange Situation at 12- and 13-months, respectively (N = 62 two-parent families). Hierarchical regression models revealed that mothers who viewed the paternal caregiving role as important were less likely to have securely attached infants, but only when infant fussiness was high. Additionally, fathers who viewed the paternal caregiving role as important were more likely to have securely attached infants, but only when infants’ fussiness or marital quality was high

Impact of oxygen levels on human hematopoietic stem and progenitor cell expansion

Oxygen levels are an important variable during the in vitro culture of stem cells. There has been increasing interest in the use of low oxygen to maximize proliferation and, in some cases, effect differentiation of stem cell populations. It is generally assumed that the defined pO2 in the incubator reflects the pO2 to which the stem cells are being exposed. However, we demonstrate that the pO2 experienced by cells in static culture can change dramatically during the course of culture as cell numbers increase and as the oxygen utilization by cells exceeds the diffusion of oxygen through the media. Dynamic culture (whereby the cell culture plate is in constant motion) largely eliminates this effect, and a combination of low ambient oxygen and dynamic culture results in a fourfold increase in reconstituting capacity of human hematopoietic stem cells compared with those cultured in static culture at ambient oxygen tension. Cells cultured dynamically at 5% oxygen exhibited the best expansion: 30-fold increase by flow cytometry, 120-fold increase by colony assay, and 11% of human CD45 engraftment in the bone marrow of NOD/SCID mice. To our knowledge, this is the first study to compare individual and combined effects of oxygen and static or dynamic culture on hematopoietic ex vivo expansion. Understanding and controlling the effective oxygen tension experienced by cells may be important in clinical stem cell expansion systems, and these results may have relevance to the interpretation of low oxygen culture studies

Accuracy of the diagnosis of malignant hyperthermia in hospital discharge records

Background: In 1997, the International Classification of Diseases, 9th Revision Clinical Modification (ICD-9CM) coding system introduced the code for malignant hyperthermia (MH) (995.86). The aim of the current study was to estimate the accuracy of coding for MH in hospital discharge records. Materials and methods: A panel of anesthesiologists expert in MH, reviewed medical records for patients with a discharge diagnosis of MH based on ICD-9 or ICD-10 codes from January 1, 2006 to December 31, 2008 at six tertiary care medical centers in North America. All cases were categorized as possible, probable, or fulminant MH, history of MH (family or personal) or other. Results: A total of 47 medical records were identified and reviewed by three experts. The mean age of patients was 40 years and 49% were male. A surgical procedure with general anesthesia was documented in 68% of patients. However, only 23.4% were judged to have had a possible, probable, or fulminant MH event. Dantrolene was given in 81% of MH cases. Family and personal history of MH accounted for 46.8% of cases. High fever without evidence of MH during admission accounted for 23.4%, and in 6.4% cases the reason for the code was not apparent. All patients judged to have an incident MH event survived to discharge. Conclusions: Medical record coding for MH typically includes both incident cases as well as a history of MH. The positive predictive value of about 70% for MH in this study are consistent with other studies of ICD-9 accuracy in the US. However, epidemiologic studies based on coded diagnosis of MH should carefully distinguish between incident cases related to anesthesia, cases unrelated to anesthesia and diagnosis based on history only

Widespread Aberrant Alternative Splicing despite Molecular Remission in Chronic Myeloid Leukaemia Patients

Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease