Do Patients Detect Changes in Breathing After Orthognathic Surgery?

BACKGROUND: Orthognathic surgery addresses facial aesthetics and function in patients with dentofacial deformities. It is associated with changes in upper airway volume (UAV). If changes in UAV are perceived by asymptomatic patients is unclear. PURPOSE: The purpose was to measure associations between changes in UAV and patient-reported benefits using patient-reported outcome measures. STUDY DESIGN: A sample presenting dentofacial deformities without reported breathing problems undergoing orthognathic surgery was retrospectively studied. Patients aged 18-30 years with 12-month follow-up were included. Patients with systemic disease, drug abuse, mental health disorder, or temporomandibular joint dysfunction were excluded. PREDICTOR: The predictor variable was changes in UAV measured in 3-dimensional computed tomography. Subjects were grouped into increased or decreased UAV. MAIN OUTCOME VARIABLE: The primary outcome variable was changes in health-related quality of life measured with Oral Health Impact Profile 49 (OHIP-49). COVARIATES: Weight, height, age, sex, and sub-scaled OHIP-49 were registered. Cephalometric measurements of hard tissue movements were recorded. ANALYSES: Mean, standard deviation, and a level of statistical significance at P < .05 were used. Differences in OHIP-49 were compared using unpaired t-test. The correlation between covariates and outcomes was analyzed using the Spearman's rank test. Analysis of covariance between the predictor and outcome, adjusted for covariates (body mass index), was performed. RESULTS: Fifty-four subjects with a mean age of 20.89 years and 52% males were enrolled. The mean change in UAV was 0.12 cm3 (standard deviation [SD] 9.21, P = .93) with a mean absolute deviation of 7.28 cm3 (SD 5.54). The mean change in OHIP-49 score was 20.93 (SD 28.90). Twenty-seven (50%) subjects had increased UAV (7.4 cm3, SD 6.13) and the other had decreased (-7.17 cm3, SD 5.01) (P = .01). At follow-up, equal levels of mean OHIP-49 score were found, but because of a baseline difference (15.74, P = .048), the subjects with and without increased UAV improved in OHIP-49 score 13.04 (SD 30.53) and 28.81 (SD 25.33), respectively (P = .04). CONCLUSIONS: Because equal levels of OHIP-49 score at follow-up, changes in UAV could not be associated with patient-reported health-related quality of life. Patient-reported outcome measure evaluations of orthognathic surgical treatment for airway obstruction should be performed in patients with a perceived impairment.

Starting a Swedish national quality registry for orthognathic surgery: a tool for auditing fundamentals of care.

BACKGROUND: National quality registries (NQRs) provide open data for user-directed acquisition. National Quality Registry (NQR) data are often used to analyze the rates of treatment success and adverse events for studies that aim to improve treatment quality and patient satisfaction. Thus, NQRs promote the goal of achieving evidence-based therapies. However, the scientific literature seldom focuses on the complex process of initiating, designing, and implementing an NQR. Starting an NQR may be particularly challenging in a setting where specialized care is decentralized, such as orthognathic surgery in Sweden. The present study describes the initiation and early phases of a new NQR for orthognathic surgery in Sweden. METHODS: The initial inventory phase included gaining knowledge on regulations, creating economic plans, and identifying pitfalls in existing NQRs. Next, a crude framework for the registry was achieved. Outcome measures were selected with a nation-wide questionnaire, followed by a Delphi-like process for selecting parameters to include in the NQR. Our inclusive process comprised a stepwise introduction, feedback-based modifications, and preparatory educational efforts. Descriptive data were collected, based on the first 2 years (2018-2019) of registry operation. RESULTS: Two years after implementation, 862 patients that underwent 1320 procedures were registered. This number corresponded to a 91% coverage rate. Bimaxillary treatments predominated, and the most common were a Le Fort I osteotomy combined with a bilateral sagittal split osteotomy (n = 275). Reoperations were conducted in 32 patients (3.6%), and the rate of patient satisfaction was 95%. CONCLUSIONS: A National Quality Registry should preferentially be started and maintained by an appointed task force of active clinicians. A collaborative, transparent, inclusive process may be an important factor for achieving credibility and high coverage, particularly in a decentralized setting.

Treatment outcomes and patient-reported quality of life after orthognathic surgery with computer-assisted 2- or 3-dimensional planning: A randomized double-blind active-controlled clinical trial.

INTRODUCTION: Thorough treatment planning is essential for a good clinical outcome in orthognathic treatment. The planning is often digital. Both 2-dimensional (2D) and 3-dimensional (3D) software options are available. The aim of this randomized 2-arm parallel double-blinded active-controlled clinical trial was to compare the outcomes of computer-based 2D and 3D planning techniques according to patient-reported health related quality of life. The hypothesis was that a 3D technique would give a better treatment outcome compared with a 2D technique. METHODS: Orthognathic treatment for 62 subjects, aged 18 to 28 years, with severe Class III malocclusion was planned with both 2D and 3D techniques. After treatment planning but before surgery, the patients were randomly allocated via blind collection of 1 enveloped card for each subject in a 1:1 ratio to the test (3D) or the control (2D) group. Thus, the intervention was according to which planning technique was used. The primary outcome was patient-reported outcome measures. The secondary outcome was relationship between patient-reported outcome measures and cephalometric accuracy. Questionnaires on the patient's health-related quality of life (HRQoL) were distributed preoperatively and 12 months after surgical treatment. The questionnaires were coded, meaning blinding throughout the analysis. Differences between groups were tested with the Fisher permutation test. The HRQoL was also compared with measurements of cephalometric accuracy for the 2 groups. RESULTS: Three subjects were lost to clinical follow-up, leaving 57 included. Of these, 55 subjects completed the questionnaires, 28 in the 2D and 27 in the 3D groups. No statistically significant difference regarding HRQoL was found between the studied planning techniques: the Oral Health Impact Profile total showed -3.69 (95% confidence interval, -19.68 to 12.30). Consistent results on HRQoL and cephalometric accuracy showed a difference between pretreatment and posttreatment that increased in both groups but to a higher level in the 3D group. A difference between pretreatment and posttreatment HRQoL was shown for both groups, indicating increased quality of life after treatment. This supports recent findings comparing 3D and 2D planning techniques. No serious harm was observed during the study. CONCLUSIONS: Improvements of HRQoL were shown after treatment independent of which planning technique, 2D or 3D, was used. No statistically significant difference was found between the planning techniques. REGISTRATION: This trial was not registered. PROTOCOL: The protocol was not published before trial commencement. FUNDING: This project was supported by personal grants to Martin Bengtsson from the Scandinavian Association of Oral and Maxillofacial Surgeons (25000 SEK), the Southern Region of the Swedish Dental Association (50000 SEK), and the Swedish Association of Oral and Maxillofacial Surgeons (25000 SEK). The sponsors had no influence on the study design, analysis of the data, or the writing of the article.

Mandibular Access Osteotomy for Tumor Ablation: Could a More Tissue-Preserving Technique Affect Healing Outcome?

PURPOSE: Paramedial mandibulotomy facilitates access for the resection of tumors in the oral and oropharyngeal space; however, severe complications related to surgical techniques and radiotherapy have been reported for this procedure. This study evaluated whether preservation of the periosteum during a mandibulotomy would decrease postoperative complications owing to the increased healing capacity provided by preserving more tissue. MATERIALS AND METHODS: Patients who underwent mandibulotomy for surgical tumor ablation from 2007 through 2012 were included in a retrospective controlled cohort study. The trial was comprised of 2 groups: 1 group underwent subperiosteal and 1 group underwent supraperiosteal surgical dissection in the area of the mandibulotomy. The primary predictor variable was surgical technique and the primary outcome variable was surgical complications. The groups were matched according to tumor type, age, and gender. Clinical and radiographic follow-up was performed 12 months after surgery. Complications regarding bone exposure, plate exposure, osteoradionecrosis, nonunion, infection of the soft tissue flap, abscesses, fistulas, and gingival necrosis were recorded. Recordings of other factors, such as age, smoking habits, and timing of radiotherapy and chemotherapy, were performed and the 2 groups were compared for postoperative complications. Data analysis used the χ(2) test (Fisher exact test) to compare the sub- and supraperiosteal groups for postoperative complications. RESULTS: Thirty-two patients were included (16 per group; 14 women and 18 men; mean age, 56 yr). Thirteen patients in the subperiosteal group and 7 in the supraperiosteal group showed complications during the first 12 months. Seven patients had complications that persisted to the 12-month follow-up (6 in the subperiosteal group and 1 in the supraperiosteal group); however, the difference was not statistically relevant. CONCLUSION: This study found more persistent complications in the subperiosteal group compared with the supraperiosteal group at 12-month follow-up, which could imply that a more tissue-preserving surgical technique promotes mandibular healing in patients undergoing mandibular access osteotomy in combination with radiotherapy.

Na+ current properties in islet α- and ß-cells reflect cell-specific Scn3a and Scn9a expression.

Mouse pancreatic ß- and α-cells are equipped with voltage-gated Na(+) currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at -100 mV and -50 mV in ß- and α-cells, respectively). Single-cell PCR analyses show that both α- and ß-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: ß-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na(+) current by 80%. In ß-cells, knockout of Scn9a lowers the Na(+) current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a(-/-) islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na(+) channel α subunit in both α- and ß-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in ß-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.

R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion.

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.

Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models.

Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023).1.

Habitat complexity affects microbial growth in fractal maze.

The great variety of earth's microorganisms and their functions are attributed to the heterogeneity of their habitats, but our understanding of the impact of this heterogeneity on microbes is limited at the microscale. In this study, we tested how a gradient of spatial habitat complexity in the form of fractal mazes influenced the growth, substrate degradation, and interactions of the bacterial strain Pseudomonas putida and the fungal strain Coprinopsis cinerea. These strains responded in opposite ways: complex habitats strongly reduced fungal growth but, in contrast, increased the abundance of bacteria. Fungal hyphae did not reach far into the mazes and forced bacteria to grow in deeper regions. Bacterial substrate degradation strongly increased with habitat complexity, even more than bacterial biomass, up to an optimal depth, while the most remote parts of the mazes showed both decreased biomass and substrate degradation. These results suggest an increase in enzymatic activity in confined spaces, where areas may experience enhanced microbial activity and resource use efficiency. Very remote spaces showing a slower turnover of substrates illustrate a mechanism which may contribute to the long-term storage of organic matter in soils. We demonstrate here that the sole effect of spatial microstructures affects microbial growth and substrate degradation, leading to differences in local microscale spatial availability. These differences might add up to considerable changes in nutrient cycling at the macroscale, such as contributing to soil organic carbon storage.

Brachytherapy and osteoradionecrosis in patients with base of tongue cancer.

BACKGROUND: Base of tongue cancer incidence and patient survival is increasing why treatment sequelae becomes exceedingly important. Osteoradionecrosis (ORN) is a late adverse effect of radiotherapy and brachytherapy (BT) could be a risk factor. Brachytherapy is used in three out of six health care regions in Sweden. AIMS: Investigate if patients treated in regions using BT show an increased risk for ORN and whether brachytherapy has any impact on overall survival. MATERIAL AND METHODS: We used data from the Swedish Head and Neck Cancer Register between 2008-2014. Due to the nonrandomized nature of the study and possible selection bias we compared the risk for ORN in brachy vs non-brachy regions. RESULTS: Fifty out of 505 patients (9.9%) developed ORN; eight of these were treated in nonbrachy regions (16%), while 42 (84%) were treated in brachy regions. Neither age, sex, TNM-classification/stage, p16, smoking, neck dissection, or chemotherapy differed between ORN and no-ORN patients. The risk for ORN was significantly higher for patients treated in brachy regions compared to non-brachy regions (HR = 2,63, p = .012), whereas overall survival did not differ (HR = 0.95, p = .782). CONCLUSIONS AND SIGNIFICANCE: Brachytherapy ought to be used cautiously for selected patients or within prospective randomized studies.

Noncovalent antibody immobilization on porous silicon combined with miniaturized solid-phase extraction (SPE) for array based immunoMALDI assays.

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 µL, 1 nM).

Single-cell gene expression profiling using reverse transcription quantitative real-time PCR.

Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.

Do patient-reported outcome measures correlate with clinical follow-up after arthroscopic treatment of internal derangement of the temporomandibular joint?

INTRODUCTION: Arthroscopic treatment of the temporomandibular joint (TMJ) has traditionally been evaluated with clinical measurements. Additional recordings of patient reported outcome measures (PROM) is expected to make the evaluation more complete. The aim of the study was to evaluate if PROM correlates with clinical follow-up measures after arthroscopic treatment of internal derangement of the TMJ. MATERIAL AND METHODS: Patients with temporomandibular dysfunction (TMD), treated with arthroscopic lysis and lavage of the TMJ, were followed with a PROM questionnaire; Jaw Functional Limitation Scale 20 (JFLS-20) and a review of patient medical records. Out of 239 patients treated, 134 were eligible for inclusion to the study. RESULTS: 91 subjects, 21 males and 70 females, with mean age 42.6 years completed the follow-up. The JFLS-20 mean score was 25.43 (range: 0-148). A correlation was found between JFLS and mouth opening. The JFLS-20 score was decreased by 1.48 (p = 0.0001) for ever millimeter larger mouth opening and with 1.16 (p = 0.001) for every gained millimeter of mouth opening after arthroscopic treatment of the TMJ. The mean maximal mouth opening was 38.43 mm preoperatively (15 to 75 mm) compared to 42.19 mm postoperatively (range: 21-75 mm). A total of 80 subjects with TMD associated pain preoperatively was reduced to 25 postoperatively (p = 0.0001). DISCUSSION: Advantageous treatment effect with arthroscopic lysis and lavage was presented. The outcome of the JFLS-20 questionnaire indicates a correlation with the clinical findings. Future studies will focus on larger cohorts and using PROM with a case-control setting.

Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.

How do fungi navigate through the complex microscopic maze-like structures found in the soil? Fungal behaviour, especially at the hyphal scale, is largely unknown and challenging to study in natural habitats such as the opaque soil matrix. We monitored hyphal growth behaviour and strategies of seven Basidiomycete litter decomposing species in a micro-fabricated "Soil Chip" system that simulates principal aspects of the soil pore space and its micro-spatial heterogeneity. The hyphae were faced with micrometre constrictions, sharp turns and protruding obstacles, and the species examined were found to have profoundly different responses in terms of foraging range and persistence, spatial exploration and ability to pass obstacles. Hyphal behaviour was not predictable solely based on ecological assumptions, and our results obtained a level of trait information at the hyphal scale that cannot be fully explained using classical concepts of space exploration and exploitation such as the phalanx/guerrilla strategies. Instead, we propose a multivariate trait analysis, acknowledging the complex trade-offs and microscale strategies that fungal mycelia exhibit. Our results provide novel insights about hyphal behaviour, as well as an additional understanding of fungal habitat colonisation, their foraging strategies and niche partitioning in the soil environment.

Habitat geometry in artificial microstructure affects bacterial and fungal growth, interactions, and substrate degradation.

Microhabitat conditions determine the magnitude and speed of microbial processes but have been challenging to investigate. In this study we used microfluidic devices to determine the effect of the spatial distortion of a pore space on fungal and bacterial growth, interactions, and substrate degradation. The devices contained channels differing in bending angles and order. Sharper angles reduced fungal and bacterial biomass, especially when angles were repeated in the same direction. Substrate degradation was only decreased by sharper angles when fungi and bacteria were grown together. Investigation at the cellular scale suggests that this was caused by fungal habitat modification, since hyphae branched in sharp and repeated turns, blocking the dispersal of bacteria and the substrate. Our results demonstrate how the geometry of microstructures can influence microbial activity. This can be transferable to soil pore spaces, where spatial occlusion and microbial feedback on microstructures is thought to explain organic matter stabilization.

Time stamped list mode data from gamma-ray spectroscopic measurements on 47 nuclear fuel assemblies performed at Clab, Sweden, September 2016 through March 2019.

Using a high-purity Germanium gamma-ray energy spectroscopic detector system, time-stamped list-mode data sets were acquired during axial scanning of 19 boiling water reactor (BWR) and 28 pressurized water reactor (PWR) type of nuclear fuel assemblies. The data sets were collected during two measurements campaigns in September 2016 and March 2019 at the Central Interim Storage Facility for Spent Nuclear (Clab) in Sweden. A certified calibration source of 137Cs was positioned along the central line of sight between the measured fuel assembly and the detector. Data sets from measurements with only the calibration source and other background sources, i.e. without a nuclear fuel assembly present, are also included. The list-mode structure of the measured data allows for an axially-resolved as well as energy-spectral resolved intensity of nuclide-specific gamma lines emitted from the spent nuclear fuel. Data presented here can be used e.g. for validation of gamma-ray transport simulation tools or for development of methods to estimate parameters of the spent nuclear fuel based on data from gamma-ray spectroscopy.

Data from calorimetric decay heat measurements of five used PWR 17x17 nuclear fuel assemblies.

Raw data from calorimetric measurements of five nuclear fuel assemblies of the PWR 17 × 17 type are provided. Measurements of the temperature both inside a calorimeter, in which the fuel assembly was placed, as well as outside, were performed as a function of time while water circulating inside the calorimeter heats up from radiation emitted in the radioactive decay of material in the fuel assembly. The data contain also measurements of dose rate in the water outside the calorimeter. Data from 38 measurements using an electrically heated model of a fuel assembly are also provided to be used for, e.g., calibration. The data can be used for validation of computer codes for modelling of nuclear systems, e.g. nuclear reactors, storage and transport of nuclear fuel or systems for geological disposal.

Assessment of Somatosensory and Psychosocial Function of Patients With Trigeminal Nerve Damage.

OBJECTIVE: The present study assessed somatosensory changes related to trigeminal nerve damage using extensive evaluation tools and assessed the effect of such damage on the patients' psychosocial status and quality of life compared with healthy participants. METHODS: In 37 patients with intraorally or extraorally presenting trigeminal nerve damage diagnosed as painful or nonpainful posttraumatic trigeminal neuropathy, psychophysical tests like quantitative sensory testing (QST) and qualitative sensory testing and the electrophysiological "nociceptive-specific" blink reflex were performed. The patients and 20 healthy participants completed a set of questionnaires assessing their psychosocial status and quality of life. RESULTS: A loss or gain of somatosensory function was seen in at least 1 QST parameter in >88.9% of the patients. Patients in whom extraoral QST was performed showed an overall loss of somatosensory function, whereas intraoral QST showed a general gain of somatosensory function. Qualitative sensory testing identified a side-to-side difference in the tactile and pinprick stimulation in >77% of the patients. An abnormal "nociceptive-specific" blink reflex response was seen in 42.1% to 71.4% of patients dependent on the trigeminal branch stimulated, though comparisons with healthy reference values showed ambiguous results. Compared with the healthy participants, patients showed higher scores for pain catastrophizing, symptoms of depression and anxiety, limited jaw function, more somatic symptoms, and significantly impaired oral health-related quality of life (all P<0.038). DISCUSSION: The results from the present study showed presence of varied somatosensory abnormalities when assessed using psychophysical and electrophysiological investigations and a significantly impaired psychosocial status.

Quantitative transcription factor analysis of undifferentiated single human embryonic stem cells.

BACKGROUND: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. METHODS: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. RESULTS: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). CONCLUSIONS: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.

Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

BACKGROUND: Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells. RESULTS: Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic beta-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined. CONCLUSION: Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.

GABA, a natural immunomodulator of T lymphocytes.

gamma-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could be activated by the low (nM-1 microM), physiological concentrations of GABA present around neurons in the brain. The cells expressed the alpha1, alpha4, beta2, beta3, gamma1 and delta GABAA channel subunits and formed functional, extrasynaptic-like GABA channels that were activated by 1 microM GABA. 100 nM and higher GABA concentrations decreased T cell proliferation. The results are consistent with GABA being immunomodulatory.