Development of an optimized conditionally replicative adenoviral agent for ovarian cancer

Human ovarian cancer is a highly lethal malignant neoplasm in woman with no effective treatment if conventional chemotherapy fails. In this regard, conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution to the development of CRAds was the introduction of tumor-selective viral replication to restrict amplification to the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells, killing the cells by cytolysis, leaving normal cells unaffected. However, to date, there have been limitations to the clinical application of these CRAd agents i.e. poor viral infectivity, poor tumor specificity and high toxicity. Here, we report the in vitro and in vivo comparison of four CRAd agents developed for ovarian cancer application, specifically, Ad-Delta24.F5/3, CRAd-C.F5/3, CRAd-M.F5/3 and CRAd-S.F5/3. All CRAd agents contained fiber knob chimeras of adenovirus serotype 3, which enhanced the viral infectivity at the transductional level via a non-Coxsackie-Adenovirus Receptor alternative pathway. In addition, these CRAds embodied distinct mechanisms for the achievement of replication specificity. Tumor cell killing was assessed by using an oncolytic assay and a cell viability assay (MTS) in vitro, while tumor growth was examined in a xenograft model in vivo by using a bioluminescent imaging assay. In addition, the replication rates of the CRAd agents were determined in human liver slices. Both the Ad-Delta24.F5/3 and CRAd-S.F5/3 were demonstrated to have higher tumor killing effects in tumor cells and a lower viral replication rate in human liver. These agents are thus excellent candidates for clinical trials of CRAd agents against human ovarian cancer.     

Low radiation dose ECG-gated chest CT angiography on a 256-slice multidetector CT scanner

Computed tomography angiography (CTA) of the thorax other than cardiac CTA, is utilized for a multitude of conditions and ranges in application from a diagnostic test, to presurgical planning and postsurgical follow-up. Helical CTA without electrocardiogram (ECG) gating has been routinely utilized for the evaluation of thoracic vasculature. However, its applicability can be limited in the evaluation of the thoracic aorta and pulmonary vasculature because of the artifacts resulting from cardiac motion. Traditional retrospective ECG-gated helical scans address this issue but at the price of a high radiation dose to the patient. In this paper we review CTA dose reduction strategies for non-coronary indications, examine field of view requirements, and discuss breath hold challenges for ECG-gated acquisitions. In addition, we present clinical examples performed using low-dose prospective gating technique for evaluation of the aorta acquired on a 256-slice multidetector computed tomography system.     

Clinical profile and distribution of peripheral retinal changes in myopic population in a hospital-based study in North India

Purpose:To evaluate the prevalence of different types of peripheral retinal changes in a myopic population in North India and correlate them with axial length.Methods:This cross-sectional, hospital-based survey included 600 eyes of 300 myopic individuals, aged between 10 and 40 years, attending the outdoor ophthalmology clinic of a tertiary eye care hospital in North India were examined from July 2019 to July 2020. They were divided into mild, moderate, high, and severe myopia according to the spherical equivalent of refraction. Axial length was recorded. Peripheral retinal changes were examined by scleral indentation binocular indirect ophthalmoscopy. Standardized findings considered with their fundus location were lattice degeneration, white without pressure and white with pressure, snail-track degenerations, peripheral chorioretinal atrophy, retinal holes, tears, and detachment. The study was approved by the institutional ethics committee, and all participants provided informed consent.Results:Peripheral retinal degenerations were found in almost half (53%) of all myopes included in the study. The most common peripheral retinal degeneration found was lattice degeneration, followed by white without pressure, white with pressure, and chorioretinal atrophy. Most of the peripheral retinal degenerations were seen in the temporal quadrant of the fundus, either superotemporal or inferotemporal. There was a significant positive association between the prevalence of peripheral retinal degeneration with age, increased axial length, and severity of myopiaConclusion:The results of our study indicate the necessity for careful peripheral fundus examinations of all myopes, irrespective of age and degree of myopia, for early diagnosis and better management of visual-threatening complications like retinal detachment.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have greatly accelerated the pace of high-resolution structure determination of biological macromolecules. However, a major bottleneck in the structural study of important targets including membrane proteins, protein–DNA complexes, and large protein assemblies is sample production. The challenge of producing functional proteins extends beyond structural biology and impacts many areas of biological sciences that currently rely on techniques of protein overexpression. Conventional methods of protein production involve overexpression of a target gene from a plasmid within heterologous systems, including bacterial (Escherichia coli), insect (Sf9), and mammalian (HEK) cells. Overexpression approaches frequently encounter issues with proteins that are misfolded, nonfunctional, or degraded by the host cells. Overexpression of certain proteins may also alter protein homeostasis within cells. Substantial optimization is often necessary to identify the right conditions to produce suitable proteins for study, which is time-consuming and challenging in many cases. The problem is compounded further by multiprotein complexes where multiple subunits need to be assembled in the correct order and stoichiometries, making the coexpression of multiple subunits and purification of protein assemblies even more challenging.Studies of challenging proteins and large protein complexes have sometimes been possible by extracting samples from natural sources. While this approach has proven successful in special cases, examples of native proteins purified in this way have been largely limited to highly abundant and large protein assemblies that can be isolated by sucrose gradient ultracentrifugation (1) or where a known binding partner can be used as bait (2, 3). A general and efficient method to isolate proteins from endogenous sources with high specificity and at sufficient quantities for structural studies remains a goal of substantial interest. A rapid and efficient approach to add an affinity tag onto a target protein in human cells for affinity purification of endogenous complexes would greatly accelerate structural characterization of challenging and biomedically important targets. Furthermore, this strategy could be combined with complementary approaches such as mass spectrometry to better understand protein function under more native-like conditions.Recent technological advances in CRISPR-Cas–mediated genome engineering have provided new avenues to target endogenous proteins from native sources by genetically incorporating an affinity tag onto a protein of interest for purification and downstream analysis (4, 5). However, the time-consuming process of generating and selecting gene-edited cells with an endogenously tagged protein has created a substantial barrier for this approach to be widely adopted by the structural biology community and other fields of protein science. A simple, fast, and minimally perturbing way to target and extract endogenous proteins would make this approach more widely accessible to the general scientific community. Here we describe a simple and highly efficient method that leverages the unique properties of split fluorescent proteins (6) to select cells harboring an affinity tag on target endogenous proteins for purification.We demonstrate this approach on the study of the human proteasome. The proteasome plays an essential role in protein degradation and is critical to maintaining cellular protein homeostasis (7). Changes to proteostasis in cells and lowered protein degradation due to decreases in proteasomal activity have been linked to aging and neurodegeneration (8). While the proteasome has been a topic of extensive studies using a variety of strategies (9–12), the study of some human proteasomal complexes has remained challenging. In this study, by tagging, purifying, and determining structures of various human proteasomal complexes from HEK cells we demonstrate that the efficient protein tagging method presented here can facilitate rapid structural studies of endogenous human protein complexes in HEK cells. Analyzing cryo-EM structures of PA28–20S complexes reveals insights into PA28 stoichiometry and sheds light on how unfolded polypeptide substrates traverse through the antechamber of the 20S core particle before entering the proteolytic chamber for degradation.     

Comparative Evaluation of Microleakage of Flowable Composite Resin Using Etch and Rinse,Self-Etch Adhesive Systems,and Self-Adhesive Flowable Composite Resin in Class V Cavities: Confocal Laser Microscopic Study

The essential factor in determining the preservation of restoration is the marginal seal. Restoring cervical lesions with a resin composite has always been a challenge. Composite resins with various viscosities and different bonding systems are being researched to reduce the microleakage. Confocal laser scanning microscopy (CLSM) is the latest non-destructive technique for visualizing the microleakage. Objectives: To evaluate and compare the microleakage of Universal Flo composite resin (G-aenial) using etch and rinse adhesive system ER-2 steps (Adper Single Bond 2), self-etch adhesive system SE-1 step (G-Bond), and self-adhesive flowable composite resin (Constic) in Class V cavities using a confocal laser scanning microscope. Materials and Method: Class V cavities were prepared on 27 caries-free human extracted premolar teeth on the buccal and lingual surfaces with standardized dimensions of 2 mm height, width 4 mm, and a depth of 2 mm. After the cavity preparation, all teeth were randomly divided into three groups, namely Group-I: G-aenial Universal Flo with Single Bond 2 (n = 9 teeth); Group-II: G- aenial Universal Flo with G-Bond (n = 9 teeth), and Group-III: Constic (n = 9 teeth). The prepared and restored specimens were then subjected to thermocycling for 500 cycles in a water bath at 5 °C and 55 °C with a dwelling time of 30 s. The specimens were placed in 0.6% aqueous rhodamine dye for 48 h. Sectioning was carried out bucco-lingually and specimens were evaluated for microleakage under a confocal laser scanning microscope. Results: There was a significant difference (p = 0.009) in microleakage when comparing total etch and rinse, specifically between Adper Single Bond 2 ER-2 steps (fifth generation) and self-adhesive flowable composite resin, which is Constic. There was more microleakage in the self-etch bonding agent, particularly G-Bond, SE-1 step (seventh generation), when compared to ER-2 steps (fifth generation bonding agent); however, the results were not statistically significant (p = 0.468). The self-adhesive flowable composite resin showed more microleakage than SE-1 step and ER-2 steps. Conclusions: None of the adhesive systems tested were free from microleakage. However, less microleakage was observed in the total etch and rinse, especially Adper Single Bond 2 (ER-2 steps), than the self-etch adhesive system SE-1 step and self-adhesive flowable composite resin. Clinical significance: Constant research and technological advancements are taking place in dentin adhesives to improve the marginal seal. This has led to the evolution of total acid-etching dentin bonding agents termed as etch and rinse (ER)-2 steps (fifth generation dentin bonding agents) and self-etching (SE) 2 steps, and SE-1 step dentin bonding agents termed as the sixth and seventh generation bonding agents, respectively.