A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.

Disruption of histone acetylation homeostasis triggers cognitive dysfunction in experimental diabetes.

Epigenetic mechanisms related to diabetes-afflicted CNS complications are largely unknown. The present study investigated the role of histone acetylation mechanisms triggering cognitive dysfunction in the Type 1 and 2 diabetic mice model. Dynamic changes in diabetic parameters like fasting blood glucose levels, glucose tolerance test, and insulin levels were observed after the induction of diabetes. Cognitive performance was significantly diminished in T1D and T2D mice examined by the Morris water maze, novel object recognition test, and Y Maze as compared to controls. Histone profiling revealed a significant reduction in H3K9/14 and H4K12 acetylation in the cortex and hippocampus of T1D and T2D mice vs Controls. While histone deacetylase (HDAC) activity was significantly elevated in brain regions of T1D and T2D mice, the histone acetyltransferase (HAT) activity remain unchanged. Significantly increased HDAC 2, HDAC 3 protein and mRNA expression observed in T1D and T2D brain regions may corroborate for increased HDAC activity. No significant change was observed in protein and mRNA expression of HDAC 1, 5, 6, and 7 in diabetic brains. Reduced H3K9/14 and H4K12 acetylation paralleled transcriptional repression of memory-related markers BDNF, SYP, and PSD-95 in the cortex and hippocampus of T1D and T2D. Pharmacological inhibition of HDAC activity by Trichostatin A enhanced the cognitive changes observed in T1D and T2D by ameliorating BDNF, SYP, Psd-95. The present study provides a better insight into molecular mechanisms related to diabetes-dependent memory changes that can help to generate new advances for therapeutics to be developed in this area.

Stapler Malfunctions in Bariatric Surgery: An Analysis of the MAUDE Database.

Background: Staple line leaks are a serious problem in bariatric surgery and a major cause of serious morbidity and mortality. Adverse events caused by medical devices are reported to the Food and Drug Administration which maintains the Manufacturer and User Facility Device Experience (MAUDE) database. We examined adverse stapler events reported to the MAUDE database, specifically with regards to bariatric surgery. Methods: The MAUDE database was queried for adverse events caused by staplers between January 1, 2018 - December 31, 2020; events reported by Intuitive, Ethicon, and Medtronic/Covidien; and limited our search to "gastric bypass", "sleeve gastrectomy", "stapler malfunction" combined with each company. Results: There were 883 adverse events reported for Medtronic, 353 for Ethicon, and 35 for Intuitive. Approximately 3.5 million staple reloads sold in the study period. The reported misfire rate for Medtronic was 0.04% and for Ethicon was 0.02%. Data for Intuitive was unavailable. The most common reported event for Medtronic was failure to fire (n = 349), followed by misfire (n = 186). For Ethicon, the most common event was failure to fire (n = 146), followed by mechanical problems (n = 27). The most common event with the Intuitive stapler was leak (n = 10) and bleeding from staple line (n = 8). Conclusions: Stapler malfunction is a very rare event in metabolic and bariatric surgery. All of the major stapler producers have transitioned to powered staplers with excellent safety profiles. Open and honest reporting about stapler malfunction is essential to determine the true safety of these ubiquitous devices.

Parathyroidectomy Versus Cinacalcet for the Treatment of Secondary Hyperparathyroidism in Hemodialysis Patients.

BACKGROUND: Secondary hyperparathyroidism in patients with end stage renal disease on dialysis is associated with bone pain and fractures in addition to cardiovascular morbidity. Cinacalcet is the most commonly used drug to treat such patients, but it has never been compared to surgery. The goal of this study is to compare the long-term outcomes and survival between cinacalcet and parathyroidectomy in the treatment of secondary hyperparathyroidism in hemodialysis patients. METHODS: Adult patients on hemodialysis who were treated with cinacalcet or parathyroidectomy in the United States Renal Data System were included. Patients treated with surgery (n = 2023) were compared using 1:1 propensity score matching ratio to a cohort of patients treated with cinacalcet. A Cox regression analysis was conducted to compare the overall mortality. RESULTS: The propensity score matching successfully created two groups with similar demographics. Patients in the surgery group had a higher mean peak PTH level prior to therapy (2066.8 vs 1425.4, P < 0.001). No difference was observed in the development of new-onset coronary artery disease (7.7% vs 7.9%, P = 0.8) or cerebrovascular disease (7% vs 6.7%, P = 0.8). Surgical patients had a higher rate of pathologic fractures (27.8% vs 24.9%, P = 0.04). Survival analysis showed that patients undergoing surgery had a better 5-year survival (65.6% vs 57.8%) and were less likely to die within the study period (HR 0.77, 95% CI 0.7-0.85, P < 0.0001). CONCLUSIONS: Patients on dialysis undergoing parathyroidectomy for the treatment of secondary hyperparathyroidism have a better overall survival than those treated with cinacalcet.

Quercetin alleviates cognitive decline in ovariectomized mice by potentially modulating histone acetylation homeostasis.

The synergism between estrogen and histone tail acetylation-mediated memory formation is not clearly understood. Here, we attempt to study the altered histone acetylation homeostasis mediated changes in cognition following ovariectomy and evaluate the protective effect of quercetin. A significant reduction in estradiol levels with subsequent depletion in spatial memory and learning functions assessed by Morris water maze, novel object recognition test and elevated plus maze was observed in ovariectomized (OVX) mice. Correspondingly, a significant decline in neuroplasticity markers like brain-derived neurotrophic factor (BDNF), synaptophysin (SYP) and postsynaptic density-95 (PSD-95) was observed in cortex and hippocampus of OVX animals. Notably, histone acetyltransferase (HAT)/histone deacetylase (HDAC) balance was significantly disrupted in cortex and hippocampus of OVX mice. Lowered extracellular signal regulated kinases (ERK) and cAMP response element binding protein activation observed in OVX brain regions might account for HAT/HDAC imbalance. Altered HAT/HDAC homeostasis results in lowered histone 3 acetylation in OVX brain that suppressed transcriptional activation of neuroplasticity-related genes. Quercetin supplementation to OVX mice for 4 weeks was able to ameliorate cognitive impairment by restoring HAT/HDAC homeostasis through ERK activation and reversing alterations in neuroplasticity markers in cortex and hippocampus of OVX mice. Taken together, our results suggest that quercetin alleviates ovariectomy-induced cognitive decline by modulating histone acetylation homeostasis.

Identification of Luminal Subtypes of Breast Carcinoma Using Surrogate Immunohistochemical Markers and Ascertaining Their Prognostic Relevance.

BACKGROUND: Therapeutic decisions in breast carcinoma are being made on the basis of tumor cell proliferation using exorbitant genomic tests. The 2013 St Gallen meeting advocated surrogate definitions for classifying tumors into luminal subtypes on the basis of immunohistochemical (IHC) markers. We studied the classification of estrogen receptor (ER)-positive tumors using these definitions as well as different methods for Ki-67 labeling index (LI) estimation. PATIENTS AND METHODS: A total of 541 ER+ invasive breast carcinoma cases from January 2012 to December 2012 were evaluated for Ki-67 LI by the average and hot spot methods. The IHC results of ER, PR, and human epidermal growth factor receptor 2 (HER2) were noted. HER2 IHC equivocal (2+) samples were subjected to HER2 fluorescence in-situ hybridization testing. Luminal subgroups created on the basis of the 2013 St Gallen meeting guidelines were correlated with clinicopathologic variables and disease-free survival. RESULTS: The distribution of luminal subtypes was as follows: luminal A-like, 13.3%; luminal B-like (HER2-), 57.9%; and luminal B-like (HER2+), 28.8%. Approximately 6% of cases were recategorized into different subgroups when the average method was used instead of the hot spot method for Ki-67 LI assessment. Younger patients (≤ 50 years), grade 3 tumors, positive axillary nodes, recurrence, and distant metastasis had a positive statistical correlation with luminal B-like (HER2-) subtype. Patients with luminal B-like (HER2-) tumors had a shorter disease-free survival compared to patients with luminal A-like tumors. CONCLUSION: Ki-67 LI, irrespective of the method of assessment, along with PR, can be efficiently used to divide ER+ tumors into prognostic subgroups in Indian patients.

Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes.

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves the timely appearance of numerous transcription factors (TFs); foremost among them, C/EBPß is expressed during the early phases of differentiation. Here, we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBPß by A-C/EBP protein inhibitor. Signals from EGFP-tagged A-C/EBP protein were observed in 3T3-L1 cells within 2 h of transfections, whereas for A-C/EBP gene transfections, equivalent signals appeared in 48 h. Following transient transfections, the expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocyte pathways were analyzed. Expectedly, the mRNA and protein expression profiles of adipocyte marker genes showed lower expression in both A-C/EBP protein- and gene-transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between A-C/EBP protein- and A-C/EBP gene-transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfrα, Pdgfrß, Ly6A, CD34, and Itgb1, showed high expression in A-C/EBP protein samples, whereas only Ly6A and CD34 were expressed in A-C/EBP gene-transfected samples. Pdgfrα and Pdgfrß, two known cell fate markers, were expressed in A-C/EBP protein-transfected samples, suggesting a possible reversal of differentiation. Our study provides evidences for the immediate and efficient knockdown of C/EBPß protein to understand time-dependent preadipocytes differentiation.

S-nitrosoglutathione prevents cognitive impairment through epigenetic reprogramming in ovariectomised mice.

The epigenetic signatures associated with cognitive decline driven by lack of estrogen in post-menopausal state, is not well-understood. The present study is an attempt to unravel the epigenetic mechanisms involved in cognitive impairment preceding ovariectomy in mice and evaluate the protective effects of S-nitrosoglutathione (GSNO). A significant decline in cognitive functions was observed in mice following ovariectomy as assessed by Morris water maze and Novel object recognition test. Administration of GSNO (100 µg/kg body weight, orally) daily for 4 weeks was found to ameliorate cognitive deficits observed in ovariectomised (OVX) mice. The activity of histone acetyl-transferase (HAT) was significantly disrupted in cortex and hippocampus of OVX mice. This was accompanied by increased activity of histone deacetylase (HDAC) and increased levels of HDAC-2, HDAC-3 causing lowered acetylated histone (H)3 levels. Reduced H3 acetylation triggers epigenetic repression of brain derived neurotrophic factor (BDNF) in cortex and hippocampus of OVX mice that may be responsible for neuronal damage and cognitive impairment. GSNO supplementation to OVX mice was able to reinstate HAT(CBP/p300) and HDAC balance through S-nitrosylation. GSNO restored histone acetylation at BDNF promoters (pII, pIV) thereby ameliorating BDNF levels and improving brain morphology and cognition. The study suggests that GSNO improves cognitive function in OVX mice by modulating epigenetic programming.

Suppression of isoproterenol-induced cardiotoxicity in rats by raspberry ketone via activation of peroxisome proliferator activated receptor-α.

The peroxisome proliferator-activated receptor-α (PPAR-α) controls the lipid and glucose metabolism and also affects inflammation, cell proliferation and apoptosis during cardiovascular disease. Raspberry ketone (RK) is a red raspberry (Rubusidaeus, Family-Rosaceae) plant constituent, which activates PPAR-α. This study was conducted to assess the cardioprotective action of RK against isoproterenol (ISO)-induced cardiotoxicity. Wistar rats were randomly divided into six groups (six rats/group). Rats were orally administered with RK (50, 100 and 200 mg/kg, respectively) and fenofibrate (standard, 80 mg/kg) for 28 days and ISO was administered (85 mg/kg, subcutaneously) on 27th and 28th day. Administration of ISO in rats significantly altered hemodynamic and electrocardiogram patterns, total antioxidant capacity, PPAR-α, and apolipoprotein C-III levels. These myocardial aberrations were further confirmed during infarct size, heart weight to body weight ratio and immunohistochemical assessments (caspase-3 and nuclear factor-κB). RK pretreatment (100 and 200 mg/kg) significantly protected rats against oxidative stress, inflammation, and dyslipidemia caused by ISO as demonstrated by change in hemodynamic, biochemical and histological parameters. The results so obtained were quite comparable with fenofibrate. Moreover, RK was found to have binding affinity with PPAR-α, as confirmed by docking analysis. PPAR-α expression and concentration was also found increased in presence of RK which gave impression that RK probably showed cardioprotection via PPAR-α activation, however direct binding study of RK with PPAR-α is needed to confirm this assumption.

A-ZIP53, a dominant negative reveals the molecular mechanism of heterodimerization between bZIP53, bZIP10 and bZIP25 involved in Arabidopsis seed maturation.

In Arabidopsis, maturation phase, an intricate process in seed formation is tightly regulated by the DNA binding activity of protagonist basic leucine zipper 53 (bZIP53) transcription factor and its heterodimerizing partners, bZIP10 and bZIP25. Structural determinants responsible for heterodimerization specificity of bZIP53 are poorly understood. Analysis of amino acid sequences of three bZIPs does not identify interactions that may favor heterodimerization. Here, we describe a designed dominant negative termed A-ZIP53 that has a glutamic acid-rich amphipathic peptide sequence attached to N-terminal of bZIP53 leucine zipper. Circular dichroism (CD) and mass spectrometry studies with equimolar mixture of three bZIP proteins in pairs showed no heterodimer formation whereas A-ZIP53 interacted and formed stable heterodimers with bZIP53, bZIP10, and bZIP25. A-ZIP53 electrostatically mimics DNA and can overcome repulsion between basic DNA binding regions of three bZIP proteins. Gel shift experiments showed that A-ZIP53 can inhibit the DNA binding of three proteins. CD studies demonstrated the specificity of A-ZIP53 as it did not interact with bZIP39 and bZIP72. Transient co-transfections in Arabidopsis protoplasts showed that A-ZIP53 inhibited three bZIPs and their putative heterodimers-mediated transactivation of GUS reporter gene. Furthermore, four newly designed acidic extensions were evaluated for their ability to interact with three bZIPs.