Two postprocessing techniques for the elimination of background autofluorescence for fluorescence lifetime imaging microscopy

The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.     

Expanding the phenotypic spectrum of TP63‐related disorders including the first set of monozygotic twins

Individuals with Tumor Protein P63 (TP63)‐related disorders are known to present with a range of phenotypic features, including ectrodactyly, ectodermal dysplasia, cleft lip/palate, Rapp‐Hodgkin, Hay–Wells, and limb‐mammary syndromes. We present six individuals from three families, including a set of monozygotic twins, with pathogenic TP63 variants who had novel clinical findings. The twins were discordant for cleft lip and palate, and the type of hand malformations, but concordant for choanal atresia, and bilateral volar nail. Both failed newborn screening for severe combined immunodeficiency (SCID) due to T‐cell lymphopenia. The second family included three family members across two generations. Two of these three family members had orofacial clefting, but the remaining child had a laryngeal web and hydrocele with no clefting or hand anomalies, highlighting the variable expressivity in TP63‐related disorders. The individual from the third family had unilateral cleft lip and palate, hydronephrosis, and bilateral volar nails. Together, these cases illustrate that: there is significant familial variability, including discordant major but concordant minor anomalies in the first ever reported set of molecularly confirmed monozygotic twins with pathogenic variants in TP63; pathogenic variants in TP63 should be considered in individuals with volar nail, which was previously only strongly associated with 4q34 deletion syndrome; and failed SCID newborn screening due to abnormal immune functioning may be part of the phenotypic spectrum of TP63‐related disorders, as it was reported in one prior individual and two of the individuals in our case series.     

Duration of injury correlates with necrosis in caerulein‐induced experimental acute pancreatitis: implications for pathophysiology

Pancreatic acinar cell necrosis is indicative of severe pancreatitis and the degree of necrosis is an index of its outcome. We studied whether the dose and duration of injury correlates with severity, particularly in terms of necrosis, in caerulein‐induced acute pancreatitis (AP) in Swiss albino mice. In addition to control group 1 (G1), groups 2 and 3 received four injections of caerulein every hour but were sacrificed at five hours (G2) and nine hours (G3) respectively, and group 4 received eight injections and was sacrificed at nine hours (G4). The severity of pancreatitis was assessed histopathologically and biochemically. The histopathological scores of pancreatitis in groups 3 and 4 were significantly higher than in groups 1 and 2 (4 vs. 1, 4 vs. 2, 3 vs. 1, 3 vs. 2; P < 0.05). TUNEL‐positive apoptotic cells were significantly higher in groups 2 and 3 compared with groups 1 and 4 (P < 0.05). Necrosis was significantly more in group 4 than other groups (37.49% (4.68) vs. 19.97% (1.60) in G2; 20.36% (1.56) in G3; P = 0.006 for G 2 vs. 4 and P = 0.019 for G 3 vs. 4). Electron microscopy revealed numerous autophagosomes in groups 2 and 3 and mitochondrial damage and necrosis in group 4. The pancreatic and pulmonary myeloperoxidase activity in group 4 was significantly higher than that in the other groups (P < 0.01). Hence, severity of pancreatitis is a function of the dose of injurious agent, while inflammation is both dose and duration dependent, which may also explain the wide spectrum of severity of AP seen in clinical practice.     

Real-world health care utilization and effectiveness of omalizumab for the treatment of severe asthma

Background

Omalizumab is indicated for the treatment of moderate to severe asthma. There is limited observational evidence on the costs and effectiveness of omalizumab.

The effects of inhaled budesonide and formoterol in combination and alone when given directly after allergen challenge

BACKGROUND: The use of combination inhaled budesonide and formoterol as maintenance and reliever therapy significantly improves the risk and the time to exacerbations in asthma. OBJECTIVES: To explore the mechanisms underlying the effect of the reliever dose on exacerbations by examining the effect of combination therapy on the allergen challenge model when given after allergen exposure. METHODS: In a randomized, double-blind crossover study, single doses of budesonide/formoterol (400/12 mug), formoterol (12 mug), budesonide (400 mug), or placebo were administered during the acute bronchoconstriction response (early airway response) immediately after allergen inhalation in 15 patients with mild asthma. Allergen-induced late airway response (LAR), sputum inflammatory markers, airway hyperresponsiveness, and exhaled nitric oxide were measured. RESULTS: All active treatments significantly attenuated the LAR, with budesonide/formoterol significantly better than its monocomponents (maximum FEV(1) fall: placebo, [mean +/- SEM] 21.2% +/- 3.1%; budesonide/formoterol, 4.2% +/- 1.4%; formoterol, 7.5% +/- 1.7%; budesonide, 10.4% +/- 1.6%). Allergen-induced change in methacholine PC(20) was significantly attenuated by budesonide/formoterol, but not by its monocomponents. Sputum cell counts and exhaled nitric oxide increased significantly after all allergen challenges, with no significant attenuation by any of the treatments. Therapy with combination and formoterol alone, but not budesonide, significantly reduced the early airway response. CONCLUSION: A single dose of budesonide/formoterol was superior to its monocomponents in attenuating the allergen-induced LAR and airway hyperresponsiveness. These effects may represent the contribution of the reliever dose to the budesonide/formoterol maintenance and reliever regimen. CLINICAL IMPLICATIONS: The protective effect against allergic airway responses with a single reliever dose of budesonide/formoterol is predominantly related to greater functional antagonism of airway smooth muscles.     

Early rapid rise in EAAT2 expression follows the period of maximal seizure susceptibility in human brain

Seizures are relatively common in the first weeks of life and can have lasting effects on brain development due to glutamate excitotoxicity. The excitatory amino acid transporter 2 (EAAT2) is responsible for the majority of glutamate uptake in the brain and mice with this gene deleted die from seizures. Therefore, we reasoned that developmental changes in the expression of EAAT2 might correlate with the period of increased susceptibility to seizures in humans, reflecting a changing vulnerability to excitotoxic insults. Expression levels of eight splice forms of EAAT2 were measured using quantitative RT-PCR from human prefrontal cortex and hippocampus at 1-2 months, 1-2 years, 8 years, 15-16 years, and 18-22 years of age. There was a significant increase in expression of most isoforms between 1-2 months and 1-2 years with isoform-specific patterns after that period. The increase in EAAT2 expression during the first 2 years of life corresponds to a period of maximal synapse formation and other changes in the glutamatergic system such as increased NMDA receptor expression. Moreover, the low expression of EAAT2 in the first months of life corresponds to the period of maximum susceptibility to seizures.     

Functional impairment in childhood OCD: development and psychometrics properties of the Child Obsessive-Compulsive Impact Scale-Revised (COIS-R).

This article documents the development, factor structure, and psychometric properties of the parent- and youth-report forms of the Child Obsessive Compulsive Impact Scale-Revised (COIS-R), a measure of obsessive-compulsive disorder (OCD)-specific functional impairment. Using a sample of 250 youth (M age = 11.7, 54% male, 80% Caucasian) diagnosed with OCD in a university hospital-based child anxiety clinic, exploratory factor analysis was employed to develop a 4-factor structure for the parent-report measure (Daily Living Skills, School, Social, Family/Activities) and a 3-factor structure for the youth-report form (School, Social, Activities). Both measures demonstrated good internal consistency, concurrent validity, and test-retest reliability. Moreover, partial correlations demonstrated significant associations between COIS-R scales and clinician global assessment of functioning scores controlling for both symptom severity and comorbid internalizing and externalizing symptomatology. These findings suggest that the COIS-R may hold utility for assessing the specific impact of OCD symptoms on youth functioning.     

A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries

The development of microwave breast cancer detection and treatment techniques has been driven by reports of substantial contrast in the dielectric properties of malignant and normal breast tissues. However, definitive knowledge of the dielectric properties of normal and diseased breast tissues at microwave frequencies has been limited by gaps and discrepancies across previously published studies. To address these issues, we conducted a large-scale study to experimentally determine the ultrawideband microwave dielectric properties of a variety of normal, malignant and benign breast tissues, measured from 0.5 to 20 GHz using a precision open-ended coaxial probe. Previously, we reported the dielectric properties of normal breast tissue samples obtained from reduction surgeries. Here, we report the dielectric properties of normal (adipose, glandular and fibroconnective), malignant (invasive and non-invasive ductal and lobular carcinomas) and benign (fibroadenomas and cysts) breast tissue samples obtained from cancer surgeries. We fit a one-pole Cole-Cole model to the complex permittivity data set of each characterized sample. Our analyses show that the contrast in the microwave-frequency dielectric properties between malignant and normal adipose-dominated tissues in the breast is considerable, as large as 10:1, while the contrast in the microwave-frequency dielectric properties between malignant and normal glandular/fibroconnective tissues in the breast is no more than about 10%.     

Oncology Residents’ Perspectives on Communication Skills and Shared Decision Making

Shared decision making (SDM) and effective communication are essential components of cancer care. Residents in oncology-related specialties were surveyed about communication skills and SDM. The response rate was 77% (17/22), and 93% stated that communication skills were very important for their specialty. Most (76%) thought their communication skills were adequate, but areas of difficulty included discussing end-of-life issues, giving hope when the prognosis was bleak and dealing with hostile patients. Only 58% of respondents had heard the term SDM, and 29% were aware of its meaning. More SDM and communication training are required for future oncology physicians.