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.

Primary Microplastics in the Ecosystem: Ecological Effects, Risks, and Comprehensive Perspectives on Toxicology and Detection Methods.

Recent discoveries of microplastics in cities, suburbs, and even remote locations, far from microplastic source regions, have raised the possibility of long-distance transmission of microplastics in many ecosystems. A little is known scientifically about the threat that it posed to the environment by microplastics. The problem's apparent size necessitates the rapid development of reliable scientific advice regarding the ecological risks of microplastics. These concerns are brought on by the lack of consistent sample and identification techniques, as well as the limited physical analysis and understanding of microplastic pollution. This review provides insight regarding some unaddressed issues about the occurrence, fate, movement, and impact of microplastics, in general, with special emphasis on primary microplastics. The approaches taken in the earlier investigations have been analyzed and different recommendations for future research have been suggested.

Exploring and analyzing the role of hybrid spectrum sensing methods in 6G-based smart health care applications.

Background: Researchers are focusing their emphasis on quick and real-time healthcare and monitoring systems because of the contemporary modern world's rapid technological improvements. One of the best options is smart healthcare, which uses a variety of on-body and off-body sensors and gadgets to monitor patients' health and exchange data with hospitals and healthcare professionals in real time. Utilizing the primary user (PU) spectrum, cognitive radio (CR) can be highly useful for efficient and intelligent healthcare systems to send and receive patient health data. Methods: In this work, we propose a method that combines energy detection (ED) and cyclostationary (CS) spectrum sensing (SS) algorithms. This method was used to test spectrum sensing in CR-based smart healthcare systems. The proposed ED-CS in cognitive radio systems improves the precision of the spectrum sensing. Owing to its straightforward implementation, ED is initially used to identify the idle spectrum. If the ED cannot find the idle spectrum, the signals are found using CS-SS, which uses the cyclic statistical properties of the signals to separate the main users from the interference. Results: In the simulation analysis, the probability of detection (Pd), probability of a false alarm (Pfa), power spectral density (PSD), and bit error rate (BER) of the proposed ED-CS is compared to those of the traditional Matched Filter (MF), ED, and CS. Conclusions: The results indicate that the suggested strategy improves the performance of the framework, making it more appropriate for smart healthcare applications.

Cost of stroke from a tertiary center in northwest India.

AIM: We aimed to study the cost of stroke, its predictors, and the impact on social determinants of the family. SETTINGS AND DESIGN: This prospective study was done in the Stroke unit and Neurology clinic between April 2009 and October 2011. MATERIALS AND METHODS: All first ever stroke patients during the study period were enrolled. Direct and indirect costs at admission, at 1 and 6 months follow-up were obtained. The follow-up included information about the patient's poststroke outcome using modified Rankin Scale (mRS), work status, modifications made at home, loan requirement, etc., RESULTS: Two hundred patients were enrolled in this study and final analysis was performed on 189 patients. The mean age was 58 ± 13 years and 128 (67.7%) were men. Majority (54%) were living in a joint family. The mean overall cost of stroke per patient was rupees (INR) 80612 at 6 months. Higher income (P = 0.008), poor outcome (mRS >2) (P = 0.001), and length of hospital stay (P = 0.001) were the cost driving factors of total cost of stroke at 6 months. There was a decline in the requirement of help (P < 0.0001) and need for loan (P = 0.003) at 6 months follow-up. CONCLUSIONS: Direct medical cost or acute care of stroke accounted for a major component of cost of stroke. Poor outcome, length of hospital stay, and higher income were the cost driving factors. The socioeconomic impact on the family decreased at follow up probably due to joint family system.

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.

Salmonella typhimurium detection and ablation using OmpD specific aptamer with non-magnetic and magnetic graphene oxide.

Foodborne diseases have increased in the last few years due to the increased consumption of packaged and contaminated food. Major foodborne bacteria cause diseases such as diarrhea, vomiting, and sometimes death. So, there is a need for early detection of foodborne bacteria as pre-existing detection techniques are time-taking and tedious. Aptamer has gained interest due to its high stability, specificity, and sensitivity. Here, aptamer has been developed against Salmonella Typhimurium through the Cell-Selex method, and to further find the reason for specificity and sensitivity, OmpD protein was isolated, and binding studies were done. Single molecular FRET experiment using aptamer and graphene oxide studies has also been done to understand the mechanism of FRET and subsequently used for target bacterial detection. Using this assay, Salmonella Typhimurium can be detected up to 10 CFU/mL. Further, Magnetic Graphene oxide was used to develop an assay to separate and ablate bacteria using 808 nm NIR where temperature increase was more than 60 °C within 30 s and has been shown by plating as well as a confocal live dead assay. Thus, using various techniques, bacteria can be detected and ablated using specific aptamer and Graphene oxide.

Adsorptive removal of methyl orange with polyaniline nanofibers: an unconventional adsorbent for water treatment.

The present study reports the adsorptive removal of methyl orange (MO) from its aqueous solution by the utilization of polyaniline (PANI) nanofibers. PANI nanofibers were synthesized by the conventional interfacial polymerization method using ammonium persulphate as an oxidant in an acidic medium. The average diameter of the synthesized PANI nanofibers was found to be 60 ± 5 nm as detected by high resolution transmission electron microscope and scanning electron microscope techniques. The synthesized PANI nanofibers were characterized by fourier transform infrared spectroscopy, X-ray diffraction, UV-VIS spectroscopy, Brunauer-Emmett-Teller surface area analyzer (BET), zero energy thermonuclear apparatus and four-probe conductivity techniques and were further used for the adsorption of the MO dye from its aqueous solution using batch mode experiments. Due to their positively charged backbone, PANI nanofibers are found to be suitable for the adsorption of anionic dyes such as MO. The BET surface area of the synthesized polyaniline nanofibers was found to be 48.83 m2 g-1 (higher than conventionally synthesized polyaniline). The influence of important adsorption parameters, i.e. initial dye concentration, pH and duration of contact was also studied, and the equilibrium values of these parameters are noted to be 7 mg L-1, pH ∼7 and 80 min, respectively. The experimental data were found to be best fitted to the pseudo-second order model (R 2 = 0.99). The Freundlich isotherm was found to be best fitted for the adsorption of MO onto PANI nanofibers with R 2 = 0.91.

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.

A rare case of trypanosomiasis in a two month old infant in Mumbai, India.

Human Trypanosomiasis is a rare occurrence in India. In the cases reported so far the disease causative species have been the species infective to animals viz., Trypanosoma lewisi and Trypanosoma evansi. These animal species usually non pathogenic in humans can acquire the desired virulence and emerge as human pathogens causing serious disease, in the right combination of environmental, host related and organism related factors. We report here a case of trypanosomiasis caused by the rodent parasite T. lewisi in a two months old infant in urban Mumbai. Under the fastly changing environmental scenario there is an urgent need to be prepared for the emerging zoonoses. Any unusual disease occurrence in a given geographical area acquires a special significance in this context and should be reported to assess its public health importance and be prepared to deal with the consequent challenges posed, if any.

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.

Detection of human papillomavirus DNA, serum p53, and p53 antibodies in patients with cervical cancer.

Human papillomavirus (HPV) was detected in 85% and 63.6% of patients with invasive cervical cancer and minor cervical abnormalities, respectively. HPV-16 was the dominant type in both groups of women. Because of the high oncogenic potential of HPV-16 and the greater chance of its persistence, a follow-up of cases with minor cervical abnormalities harboring HPV-16 is warranted in order to observe the progression of the lesion. As many as 61.5% of the cases with invasive cervical cancer were found to have higher levels of serum p53 protein than did healthy controls. None of the patients had antibodies against the overexpressed p53. This suggests that, even if mutated, the p53 protein may not be immunogenic in all cases. An inverse relationship between the presence of HPV and the alteration in p53 expression was observed in 71.43% of the cases. This could mean the loss of p53 function as a result of either HPV-E6-mediated degradation or mutation in the p53 gene.

An investigation into the JE epidemic of 2000 in Upper Assam--a perspective study.

The viral encephalitis epidemic during 2000 involved all the seven districts of Upper Assam (with 96.4% cases). The epidemiological, serological, clinical and entomological observations of the epidemic confirmed it to be due to JE. These were analysed, presented and discussed in the light of past history/records of the disease in the state. The epidemic was confined to the peak monsoon months of July and August (96% of the cases in the past). Serologically, 75-90% cases were confirmed to be due to JE. Case fatality Rate was 42.11% (overall average of the state in the past). There was no difference in case incidence among children or adults. Male to female ratio was 1:0.6. Based on the abundance and host preferences during the transmission period as well as virus isolation studies (as per past studies in the area) the main vectors Culex vishnui and Mansonia annulifera were susceptible to 4% DDT.

Cost of stroke in low and middle income countries: a systematic review.

BACKGROUND: There is limited information available from low and middle-income countries regarding the cost of stroke treatment. Hence, we aimed to review the costs of stroke in low and middle-income countries. SUMMARY OF REVIEW: The literature search was done using databases: PubMed/Medline, Ovid, EconLit and Google Scholar from 1966 until January 2014 using key words 'economic impact of stroke', 'cost of illness in low and middle income countries' and 'cost of stroke'. With these key words, 10/184 articles were retained for this review. The cost data were converted to 2013 currency values using a web-based tool (CCEMG-EPPI-centre cost converter). Most of the included studies were conducted in Asia. The design of these studies was retrospective and conducted predominantly in multicenter private hospitals. The highest mean direct medical cost of stroke was US$ 8424 in Nigeria. The lowest mean cost of stroke was in Senegal (US$ 416). The average length of hospital stay was longest (20 days) in China. The main predictors of higher costs appeared to be due to differences in length of stay and stroke severity. CONCLUSION: Costs of stroke are variable because of heterogeneous healthcare systems prevailing in low and middle-income countries. Length of hospital stay and stroke severity appear to be the main predictors of cost. Understanding the costs of stroke in low and middle-income countries is important. However, the evidence remains limited because there is a lack of standardized research. Future research should focus on using a uniform method across low and middle-income countries for estimating the costs of stroke.