Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and NaV1.5 blockade

Introduction

The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa_V1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture.

Methods

Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds.

Results

Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC₅₀s (paired values denote constantly cultured and frozen, respectively) of 4.8 ± 0.4 and 5.1 ± 0.4, 1.4 ± 0.1 and 1.1 ± 0.1, 24.4 ± 2.4 and 21.9 ± 1.8, 2.1 ± 0.4 and 2.1 ± 0.1, 5.2 ± 0.4 and 4.0 ± 0.2 μM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa_V1.5 with IC₅₀s of 46.6 ± 4.3 and 28.0 ± 2.3, 7.6 ± 0.7 and 6.2 ± 0.5, 153.5 ± 13.0 and 106.0 ± 4.7, 5.5 ± 0.5 and 4.8 ± 0.2 μM. Voltage dependences of activation (V_1/2) for hERG were statistically identical, 0.4 ± 0.8 mV and 2.5 ± 0.5 mV. In hNa_V1.5, the V_1/2 of inactivation and activation were statistically identical, −82.7 ± 0.1 mV versus − 84.9 ± 0.3 mV, −47.5 ± 0.3 mV versus − 45.0 ± 0.6 mV. Current density in both conditions in hERG experiments was similar, 47.0 ± 4.1 pA versus 42.3 ± 6.0 pA/pF.

Discussion

hERG and hNa_V1.5 screens run using frozen cells have statistically identical IC₅₀s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.     

Rhinocerebral mucormycosis: the disease spectrum in 27 patients

The variable forms of clinical complaints, findings and time interval of presentation in 27 cases of mucormycosis have been described, which were encountered over a span of 8 years. The previous concept about this fungal infection attacking chronic, debilitated, immunocompromised patients does not appear to hold true. Seven of the 27 patients (22.2%) did not reveal any predisposing factors and their outcome of 42.9% survival seems to be poorer than the total outcome (66.7%). 'Chronic form' of disease presentation, the definition of which is still not delineated, was encountered in four patients (14.8%). Again, the outcome was not significantly different from the total survival. Burr-hole tap of an intracranial abscess revealing mucor in a 2-month-old infant has been described. Even in the present era, extranasal exenteration of sinuses along with disfiguring orbital exenteration is required to ensure satisfactory surgical debridement. Control of the underlying predisposing illness, along with the aggressive surgical debridement and the parenteral administration of amphotericin B, remains the treatment essentials even today.     

Effects of stimulus structure and target-distracter similarity on the development of visual memory representations in schizophrenia

Introduction. In a previous study (Silverstein, Bakshi, Chapman, & Nowlis, 1998a, Cognitive Neuropsychiatry) we demonstrated that while schizophrenia patients showed similar learning curves as nonpatient controls when determining whether a configural pattern of elements has been seen before or not, they did not demonstrate any learning curve with nonconfigural stimuli (in contrast to controls). Methodological limitations of that study, however, precluded generalisability of those effects.Methods. In the present study, therefore, different groups of schizophrenia patients (n = 18) and controls (n = 22) were administered a modified version of the familiarity judgement task used in Silverstein et al. (1998a). In the new version of the task: (1) the patterns that repeated were different for each participant; and (2) half of the nonrepeating patterns were configural and half were nonconfigural.Results. These indicated stronger perceptual learning effects for the configural compared to the nonconfigural patterns, and overall better performance and more pronounced learning effects for the control group.Conclusions. The data provide further evidence for a schizophrenia-related impairment in perceptual organisation and in the ability to develop memory representations for novel stimuli.     

Nutrition assessment in patients undergoing liver transplant

Liver transplantation (LT) is a major surgery performed on patients with end stage liver disease. Nutrition is an integral part of patient care, and protein-energy malnutrition is almost universally present in patients suffering from liver disease undergoing LT. Nutrition assessment of preliver transplant phase helps to make a good nutrition care plan for the patients. Nutrition status has been associated with various factors which are related to the success of liver transplant such as morbidity, mortality, and length of hospital stay. To assess the nutritional status of preliver transplant patients, combinations of nutrition assessment methods should be used like subjective global assessment, Anthropometry mid arm-muscle circumference, Bioelectrical impedance analysis (BIA) and handgrip strength.     

Unusual presentation of laryngeal foreign bodies — report of two rare cases

Aspiration of a foreign body into the respiratory tract is a common and serious problem in childhood but is rare in adults. Two interesting cases in which the diagnosis of foreign body in the larynx was not suspected preoperatively are being reported. A three and half year old female child was diagnosed as a case of bronchitis and was later found to have a metallic spring in the supraglottic region which was removed by direct laryngoscopy under general anesthesia. The other patient, a 32-year-old male, was diagnosed to have a fibrous lesion in the larynx one year previously, and on laryngoscopy a vegetable, spiculated foreign body was found after excising the fibrous lesion on the left false cord. Both the cases are symptom free 18 months after endoscopy.     

MTHFR Gene Polymorphisms and the Risk of Acute Lymphoblastic Leukemia in Adults and Children: A Case Control Study in India

Genetic polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) gene have been associated with the development of acute leukemias and various malignancies. The role of MTHFR polymorphism in the development of pediatric acute lymphoblastic leukemia (ALL) has been extensively studied among north Indians in various settings, yet its association with acute leukemias remains unresolved. To evaluate the relationship between functional MTHFR polymorphisms, C677T and A1298C and possible effect on risk of ALL in adults and children in North Indian population by comparing them with healthy controls. DNA was isolated from peripheral blood of 184 ALL patients (33 adults, 151 children) and 155 controls and analyzed by a PCR-restriction fragment length polymorphism assay. The frequency of MTHFR 677CT and 1298 AC genotypes were significantly lower among adult ALL cases when compared to the controls. We found a 1.74-fold reduced risk of ALL in individuals with 1298AC polymorphic variant and a 9.17-fold decreased risk of adult ALL. However, no statistically significant difference was evident between the above polymorphisms and susceptibility to ALL in children. Polymorphisms in the MTHFR gene possibly modulate risk of ALL in north Indian adults but not in children, although larger studies are needed.     

Bone marrow transplantation for adolescents and young adults with sickle cell disease: Results of a prospective multicenter pilot study

We conducted a multicenter pilot investigation of the safety and feasibility of bone marrow transplantation (BMT) in adults with severe sickle cell disease (SCD) (NCT 01565616) using a reduced toxicity preparative regimen of busulfan (13.2 mg/kg), fludarabine (175 mg/m²) and thymoglobulin (6 mg/kg) and cyclosporine or tacrolimus and methotrexate for graft-vs-host disease (GVHD) prophylaxis. Twenty-two patients (median age 22 years; range 17-36) were enrolled at eight centers. Seventeen patients received marrow from an HLA-identical sibling donor and five patients received marrow from an 8/8 HLA-allele matched unrelated donor. Before BMT, patients had stroke, acute chest syndrome, recurrent pain events, were receiving regular red blood cell transfusions, or had an elevated tricuspid regurgitant jet (TRJ) velocity, which fulfilled eligibility criteria. Four patients developed grades II-III acute GVHD (18%) and six developed chronic GVHD (27%) that was moderate in two and severe in one patient. One patient died of intracranial hemorrhage and one of GVHD. Nineteen patients had stable donor chimerism, 1-year post-transplant. One patient who developed secondary graft failure survives disease-free after a second BMT. The one-year overall survival and event-free survival (EFS) are 91% (95% CI 68%-98%) and 86% (95% CI, 63%-95%), respectively, and 3-year EFS is 82%. Statistically significant improvements in the pain interference and physical function domains of health-related quality of life were observed. The study satisfied the primary endpoint of 1-year EFS ≥70%. This regimen is being studied in a prospective clinical trial comparing HLA-matched donor BMT with standard of care in adults with severe SCD (NCT02766465).