Enhanced Precision of Circular Dichroism Spectral Measurements Permits Detection of Subtle Higher Order Structural Changes in Therapeutic Proteins

Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision. Moreover, once spectral changes are detected, interpreting the differences in terms of specific structural attributes is challenging. Spectral normalization by the protein concentration remains one of the largest sources of error and reduces the ability to confidently detect differences in CD spectra. This work develops a simple method to enhance the precision of the CD spectral measurements through normalization of the CD spectra by the protein concentration determined directly from the CD measurement. This method is implemented to successfully detect small CD spectral changes in multiple forced degradation studies as well as comparability assessments during biologics drug development. Furthermore, the interpretation of CD spectral changes in terms of HOS differences are provided based on orthogonal data in conjunction with structural insights gained through in silico homology modeling of the protein structure.     

Protection against myocardial ischemia-reperfusion injury by the angiogenic Masterswitch protein PR 39 gene therapy: the roles of HIF1alpha stabilization and FGFR1 signaling

PR-39, a proline-arginine-rich angiogenic response peptide, has been implicated in myocardial ischemic reperfusion injury. The present study examined the cardioprotective abilities of PR39 gene therapy. Male C57Bl/J6 mice were randomized to intramyocardial injecton of 10(9) p.f.u. adenovirus encoding PR39 (PR39), FGFR1 dominant negative signaling construct (FGFR1-dn), empty vector (EV), or PR39 adenovirus plus 4 microg of plasmid endcoding a HIF1alpha dominant negative construct (PR39 + HIF1alpha-dn). Seven days later, hearts were subjected to 20 min of ischemia (I) and 2 h. reperfusion (R) ex vivo and aortic and coronary flow, left ventricular developed pressure (LVDP), and LVdp/dt were measured. Myocardial infarct (MI) size and cardiomyocyte apoptosis were measured by TTC staining and TUNEL, respectively. PR39 expression was robust up to 14 days after gene transfer and was absent after EV and FGFR1-dn. Hemodynamics showed no differences at baseline, and heart rate remained unchanged in all groups throughout the experiment. After I-R, hemodynamics remained unchanged in PR39 hearts, but deteriorated significantly in the other groups, except for aortic flow, which remained significantly higher in FGFR1-dn than in EV and PR39 + HIF1alpha-dn (p < 0.05), although it was lower than in PR39 (p < 0.05). MI was 8.7 +/- 0.9 % in PR39, 23.8 +/- 1.1% in FGFR1-dn, 29.9 +/- 2.2% in EV, and 30.8 +/- 2.7 % in PR39 + HIF1alpha-dn (PR39 vs. other groups: p < 0.05; FGFR1-dn vs. EV and PR39 + HIF1alpha-dn: p < 0.05). In PR39, HIF-1alpha protein was higher than in FGFR1-dn and EV. Importantly, cotransfection of HIF1alpha-dn with PR39 completely abolished cardioprotection by PR39. Cardioprotection by PR39 is likely conveyed by protective metabolic and survival responses through HIF1-alpha stabilization and not by angiogenesis, because baseline coronary flow was the same in all groups. Abrogation of FGFR1 signaling conveyed an intermediate degree of cardioprotection.     

A theranostic nanocomposite system based on iron oxide-drug nanocages for targeted magnetic field responsive chemotherapy

In this work, a theranostic nanocage system was developed for the targeted delivery of the anti-cancer agents camptothecin (CPT) and luotonin A (LuA). The core of the nanocage system (Fe₃O₄@OA-AD-SP NCs) was formed by biogenically synthesized Fe₃O₄ nanoparticles (NPs) decorated with a model anti-cancer drug (AD) and biosurfactant saponin (SP). The Fe₃O₄@OA-AD-SP NCs showed a high lipophilic AD loading efficiency (>80%) and a controlled pH-responsive drug release in stimulated cancerous cells in pH 6.4 media buffer. In addition, Fe₃O₄@OA-AD-SP NCs exhibited better serum protein binding efficacy at physiological pH values (7.4), furthering the important role of SP surface decoration. Particularly, these NCs showed better chemotherapeutic efficacy when examined in MCF-7 and HeLa cancer cell lines with a specific targeting capacity. Therefore, this study provides a new nano platform based on magnetic targeting and pH responsive lipophilic anticancer drug delivery to the cancer site.     

Is preoperative histological diagnosis necessary for cholangiocarcinoma?

Surgery is currently the only curative treatment for patients with cholangiocarcinoma (CCA). Whether histological diagnosis of CCA is necessary before surgery is controversial. Fifteen percent of patients with suspected biliary malignancy who undergo surgery are found to have benign disease. Surgery is a major procedure with significant morbidity and mortality and alternative treatment is available for those known to have benign stenoses. The aim of this review was to determine whether any of the current diagnostic tests have sufficient sensitivity and specificity to identify patients with benign and malignant bile duct stenoses. A literature search was performed until July 2007 to obtain information from studies published in the previous 10 years. Only studies reporting an appropriate reference test (confirmation of malignancy by biopsy, confirmation of benign nature by histology following surgical excision, or at least 6 months of follow-up for all patients) were included for review. The diagnostic odds ratio was used to measure diagnostic performance. Forty-one references of 34 studies were included in this review. None of the studies used differential verification. Six studies used blinding of assessor. None of the diagnostic tests had sufficient diagnostic accuracy to reliably separate patients with benign from malignant biliary strictures. Differentiating benign from malignant bile strictures is an important aim. There is no trial evidence demonstrating benefit in obtaining a preoperative histological diagnosis of CCA. New methods are required for stricture assessment.     

A Detailed Protocol for Generation of Therapeutic Antibodies with Galactosylated Glycovariants at Laboratory Scale Using In-Vitro Glycoengineering Technology

N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG.     

Density and Fat Fraction of the Psoas,Paraspinal, and Oblique Muscle Groups Are Associated With Lumbar Vertebral Bone Mineral Density in a Multi-Ethnic Community-Living Population: The Multi-Ethnic Study of Atherosclerosis

Low vertebral bone mass is a major risk factor for vertebral compression fractures. Although sarcopenia has been shown to be associated with low bone mineral density (BMD), it is not known whether trunk musculature is directly associated with lumbar BMD, and whether exercise modifies this association. Using data from the Multi-Ethnic Study of Atherosclerosis (MESA), we sought to determine the association of muscle density and fat fraction of the psoas, paraspinal, and oblique muscle groups with L₃ lumbar volumetric BMD, and whether these associations were modified by exercise. We obtained L₃ vBMD measurements, and fat and muscle measurements (in Hounsfield units [HU]) from abdominal computed tomography (CT) scans spanning the L₂–L₄ intervertebral disc spaces. Muscle density was defined as the mean HU value for a muscle group area. Fat fraction was calculated as the mean HU value for the muscle group fat area/total muscle group area (cm²). Exercise data were self-reported (MET-minute/week). We utilized multivariable linear regression to evaluate these associations, stratified by gender, and adjusting for demographics, body mass index (BMI), smoking status, impaired fasting glucose, and corticosteroid and anti-resorptive medication use. Among 1923 MESA participants, mean ± standard deviation (SD) age was 62 ± 10 years, 49% were female, 40% white, 21% black, 26% Hispanic/Latino, and 13% Chinese. In fully adjusted analysis, for every 1-SD higher psoas fat fraction, there was a 3.19-SD lower L₃ vBMD in men and 4.3-SD lower L₃ vBMD in women (p < 0.001). For every 1-SD higher psoas density, there was a 0.2-SD higher L₃ vBMD (p < 0.001) in men and 0.19-SD higher L₃ vBMD (p < 0.001) in women. Findings were similar for paraspinal and oblique muscles. Intentional exercise did not modify these associations. In men and women, trunk muscle density was positively associated with higher lumbar BMD, suggesting a local association. Future studies are warranted to determine the temporality of this association. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Etiology and risk factors associated with a pruritic papular eruption in people living with HIV in India

Introduction

Papulopruritic eruption (PPE) occurs in people living with HIV in India. Understanding the risk factors associated with this disease may help decrease the prevalence of PPE.

Methods

This study was a case-control study performed at the Government Hospital of Thoracic Medicine, a tertiary care hospital in Chennai, India. Cases included HIV-positive, antiretroviral (ARV) therapy-naïve adults experiencing a pruritic skin eruption for longer than one month, with evidence of multiple papular or nodular lesions and biopsy consistent with arthropod bite. Controls included HIV-positive, ARV-naïve patients without active skin rash. Main outcome measures were CD4 cell count, histology, and environmental exposures. We performed statistical analysis using Epi Info version 3.5.1 and SPSS version 11.0 (SPSS Inc., Chicago, IL). Categorical variables such as gender, urban versus rural residence, occupation, treatment history, CD4 count, use of insect repellents, and environmental exposures were evaluated using the χ² test (or the Fisher exact test when an expected value for a category was less than 5). The t-test was used to evaluate differences in age and the duration since HIV diagnosis. The Mann-Whitney test was used to compare non-normally distributed values such as CD4 cell count. A p-value that was less than 0.05 was considered to be statistically significant.

Results

Forty-one cases and 149 control subjects were included. Subjects with PPE had significantly lower CD4 cell counts compared to controls (225.5 cells/µL vs. 425 cells/µL; p=0.0001). Sixty-six percent of cases had a CD4 cell count less than 350 cells/µL. PPE cases were less likely to use mosquito repellent techniques (odds ratio 2.81, CI = 1.45–5.45).

Discussion

PPE may be an altered and exaggerated immune response to arthropod bites in HIV-positive patients. CD4 cell count is significantly lower in patients with PPE, and therefore it may be considered a qualifying clinical finding for ARV initiation in resource-poor settings. Protective measures against mosquito bites appeared to be important in preventing PPE in subjects at risk.     

Role of Immunohistochemistry in Acute Leukemias with Myelonecrosis

Myelonecrosisis a rare antemortem finding most commonly seen in haematopoeitic neoplasms, especially in acute leukemia. When myelonecrosis occurs at the time of presentation, it imposes certain diagnostic issues in sub categorization of leukemias which is necessary for therapeutic as well as prognostic purposes. Flow cytometry, though is a powerful modality, has its own limitations especially when the cells are not fresh and viable; and when the specimen is not of adequate cellularity which is usual in cases of myelonecrosis. In such situations, immunocytochemistry (ICC) or immunohistochemistry (IHC) may play a major role in lineage specification in leukemias as the necrosed marrow with the ghost cells can still retain the antigenicity for certain immunomarkers. Four such interesting cases of common B acute lymphoblastic leukemia (ALL) where IHC was used for diagnosis were included. ICC and IHC done on the necrosed marrow contributed to the diagnosis of ALL in all the four cases and contributed to subsequent management. ICC and IHC if contributory can play a major role in identifying the primary cause of myelonecrosis as the ghost cells can retain the antigenicity despite being morphologically non-viable.     

Endoscopic cartilage versus temporalis fascia grafting for anterior quadrant tympanic perforations — A prospective study in a tertiary care hospital

Objective

Management of anterior perforations of tympanic membrane is a surgical challenge. The objective of this study is to analyse and compare the results of composite cartilage perichondrium island (CCPI) graft and temporalis fascia graft by endoscopic technique in anterior quadrant perforations.