Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.

The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.

Association of mutation and expression of the brother of the regulator of imprinted sites (BORIS) gene with breast cancer progression.

INTRODUCTION: The BORIS, 11 zinc-finger transcription factors, is a member of the cancer-testis antigen (CTA) family. It is mapped to chromosome number 20q13.2 and this region is genetically linked to the early onset of breast cancer. The current study analyzed the correlation between BORIS mutations and the expression of the protein in breast cancer cases. MATERIALS AND METHODS: A population-based study including a total of 155 breast cancer tissue samples and an equal number of normal adjacent tissues from Indian female breast cancer patients was carried out. Mutations of the BORIS gene were detected by polymerase chain reaction-single standard confirmation polymorphisms (PCR-SSCP) and automated DNA sequencing and by immunohistochemistry for BORIS protein expression were performed. The observed findings were correlated with several clinicopathological parameters to find out the clinical relevance of associations. RESULTS: Of all the cases 16.12% (25/155) showed mutations in the BORIS gene. The observed mutations present on codon 329 are missense, leading to Val> Ile (G>A) change on exon 5 of the BORIS gene. A significant association was observed between mutations of the BORIS gene and some clinicopathological features like nodal status (p = 0.013), estrogen receptor (ER) expression (p = 0.008), progesterone receptor (PR) expression (p = 0.039), clinical stage (p = 0.010) and menopausal status (p = 0.023). The protein expression analysis showed 20.64% (32/155) samples showing low or no expression (+), 34.19% (53/155) with moderate expression (++), and 45.17% (70/155) showing high expression (+++) of BORIS protein. A significant association was observed between the expression of BORIS protein and clinicopathological features like clinical stage (p = 0.013), nodal status (p = 0.049), ER expression (p = 0.039), and PR expression (p = 0.027). When mutation and protein expression were correlated in combination with clinicopathological parameters a significant association was observed in the category of high (+++) level of BORIS protein expression (p = 0.017). CONCLUSION: The BORIS mutations and high protein expression occur frequently in carcinoma of the breast suggesting their association with the onset and progression of breast carcinoma. Further, the BORIS has the potential to be used as a biomarker.

Cross-sectional community-based study in Saudi Arabian population with inclusion of behavioral studies of osteoporosis patients.

Purpose: Osteoporosis is especially prevalent in the older population and in women. Fractures lead to many health complications, placing an enormous burden on the health system. Improved knowledge will encourage healthy practices that prevent osteoporosis and help community members avoid unhealthy lifestyles and habits. The goal of this study was to evaluate the knowledge, attitudes, and practices relating to osteoporosis among male and female students of the Jazan community. Methods: The Jazan region has 13 governorates and a population of more than 1.5 million. For this study, 440 participants were selected from seven different governorates, and the osteoporosis knowledge assessment tool (OKAT) was used together with five background variables and some questions on attitude and practice. A Chi-squared test was implemented to identify any significant relationships between the background variables and the knowledge and practice scores. A P value of less than 0.05 was considered significant. Results: Of the 440 participants, 53% were men and 47% women. Thirty-three percent of them held government jobs, 73% had completed university, and 50% had a monthly income of less than 5,000 riyals. Regarding the risk factors for osteoporosis, 62 (14%) smoked, 37 (8.4%) used steroids, 184 (41.8%) had a family history of osteoporosis, and 393 (89.3%) had not attended an education campaign on osteoporosis. The highest number of correct answers in the OKAT was to the question "osteoporosis increases the risk of fractures" (423, 96.1%), and the lowest number of correct answers was to the question "osteoporosis is more common in men" (60, 13.6%). Regarding protective behaviors against osteoporosis, namely physical activity, daily intake of dairy products, attending an education campaign, and background characteristics, attending an education campaign was statistically significant, with a P value of 0.003, but marital status, education level, body mass index (BMI), and occupation were not statistically significant. For the risk factors, sex was statistically significant, along with the daily soft drink intake and smoking but not with the use of steroids. Other background characteristics, such as education level and BMI, were not statistically significant as osteoporosis risk factors. Conclusions: The participants' level of knowledge was high, and the risk factors for osteoporosis were still present together with a low level of achievement for protective measures. It is highly recommended that education campaigns be implemented to increase knowledge and encourage improved attitudes and practices.

Wear and Color Stability of Preheated Bulk-fill and Conventional Resin Composites.

OBJECTIVES: Bulk-fill resin composite is commonly used in direct restorations. It is recommended that the high-viscosity version of these materials be preheated to improve flowability and adaptability. It is unknown what effects preheating (PH) might have on the wear resistance and color stability of these resin composites. This study compared the wear and color stability of high-viscosity bulk-fill and conventional resin composites with and without PH, using the CIEDE2000 formula. METHODS AND MATERIALS: Thirty-two disc-shaped specimens (n=16, 10×3 mm) were prepared with Filtek One Bulk Fill Restorative (FOBFR; 3M ESPE) and Filtek Supreme Ultra (FSU, 3M ESPE) to determine wear. Each group was divided into subgroups based on preparation conditions, that is, PH and room temperature (RT) (n=8). Thermomechanical fatigue was applied to specimens, and volumetric material loss was calculated using a laser scanner (LAS-20, SD Mechatronik, Feldkirchen-Westerham, Germany) and three-dimensional imaging software (Geomagic, 3D Systems, Rock Hill, SC, USA). An additional 124 disc-shaped specimens (n=64, 10×1 mm) were prepared with FOBFR and FSU, and subgroups were formed based on preparation conditions (PH and RT, n=32). Specimens were aged for 14 days in coffee, grape juice, distilled water, and thermocycling (20,000 cycles, 5°C-55°C). To determine color change, the lightness (L), chroma (C), and hue (H) values were measured at baseline and after 14 days using the CIEDE2000 formula. All data were statistically analyzed, and a 95% significance level was set. RESULTS: In both the PH and RT groups, FSU exhibited greater wear than FOBFR (p<0.05). Both materials were subjected to increased wear as a result of PH. After aging, a significant difference in color change was observed (p<0.05) when controlling for time and resin composite type, except for deionized water and thermocycling (p>0.05). PH had no effect on the color stability of the resin composite at any time (p<0.05). CONCLUSION: The high-viscosity bulk-fill resin composite displayed superior wear resistance and similar color stability compared with its conventional counterpart. PH resin composite may increase its susceptibility to wear and has no effect on its color stability.

Epigenetic Alteration and its Association With Downregulated FOXP3 Gene in Indian Breast Cancer Patients.

Background: FOXP3 gene, known to be a potential tumor suppressor, has been identified to interact with HER2 in mammary cancer. Moreover, the high expression of FOXP3 serves as a good predictor of the survival of patients in breast cancer, prostate cancer, and gastric cancer. The expression and epigenetic alterations were evaluated in female breast cancer patients. Material and Methods: Expression studies at the mRNA level and protein level were conducted in 140 breast cancer cases by real-time PCR and immunohistochemistry, respectively. Epigenetic studies were also conducted by analyzing the methylation status at the promoter region of the gene using MS-PCR. Results: FOXP3 mRNA expression and protein expression were downregulated in breast cancer patients. The absence of FOXP3 protein expression is significantly associated with promoter methylation, where 70 methylated cases exhibited protein loss (70/95, 73.6%). Statistically, we also found a significant correlation between FOXP3 protein expression and TNM stage, promoter methylation, and histological grade. The methylated FOXP3 cases that did not express protein were also significantly associated with positive lymph node metastasis and HER-2 status. Conclusion: The expression profile of FOXP3 may serve as a prognostic factor. In short, FOXP3 may stand in the most crucial list of biomarkers for breast cancer, bringing compelling results in terms of treatment and management of the disease.

Effect of Preheating and Fatiguing on Mechanical Properties of Bulk-fill and Conventional Composite Resin.

CLINICAL RELEVANCE: Bulk-fill composite resins may have comparable mechanical properties to conventional composite resin. Preheating does not reduce the mechanical properties of composite resins.

Effect of Resin-modified Glass Ionomer Cement Dispensing/Mixing Methods on Mechanical Properties.

STATEMENT OF THE PROBLEM: Resin-modified glass ionomer cements (RMGIs) are often used for luting indirect restorations. Hand-mixing traditional cements demands significant time and may be technique sensitive. Efforts have been made by manufacturers to introduce the same cement using different dispensing/mixing methods. It is not known what effects these changes may have on the mechanical properties of the dental cement. PURPOSE: The purpose of this study was to evaluate the mechanical properties (diametral tensile strength [DTS], compressive strength [CS], and fracture toughness [FT]) of RMGIs with different dispensing/mixing systems. METHODS AND MATERIALS: The RMGI specimens (n=14)-RelyX Luting (hand mix), RelyX Luting Plus (clicker-hand mix), RelyX Luting Plus (automix) (3M ESPE), GC Fuji PLUS (capsule-automix), and GC FujiCEM 2 (automix) (GC)-were prepared for each mechanical test and examined after thermocycling (n=7/subgroup) for 20,000 cycles to the following: DTS, CS (ISO 9917-1) and FT (ISO standard 6872; Single-edge V-notched beam method). Specimens were mounted and loaded with a universal testing machine until failure occurred. Two-/one-way analysis of variance followed by Tukey honestly significantly different post hoc test was used to analyze data for statistical significance ( p<0.05). RESULTS: The interaction effect of both dispensing/mixing method and thermocycling was significant only for the CS test of the GC group ( p<0.05). The different dispensing/mixing methods had no effect on the DTS of the tested cements. The CS of GC Fuji PLUS was significantly higher than that of the automix version ( p<0.05). The FT decreased significantly when switching from RelyX (hand mix) to RelyX Luting Plus (clicker-hand mix) and to RelyX Luting Plus (automix) ( p<0.05). Except in the case of the DTS of the GC group and the CS of GC Fuji PLUS, thermocycling had a significant effect reducing the mechanical properties of the RMGI cements ( p<0.05). CONCLUSIONS: Introducing alternative dispensing/mixing methods for mixing RMGIs to reduce time and technique sensitivity may affect mechanical properties and is brand dependent.

A case of primary diffuse large B-cell non-Hodgkin's lymphoma misdiagnosed as chronic periapical periodontitis.

Lymphoma is a malignant neoplasm of component cells of the lymphoid system which is very rare in the jaws. Here we report a case of primary diffuse large B-cell lymphoma located in the periapical region of a mandibular molar which was misdiagnosed as chronic periapical periodontitis. The present case was diagnosed at an early stage and effectively managed by chemotherapy. Although lymphoma of the mandible is rare, it must be considered in the differential diagnosis of radiolucent lesions in this region. Lack of knowledge of this rare presentation may lead to delays in diagnosis and poor prognosis.