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.

A review study on forensic optometry: new insights in forensic science to solve crime scenes, domestic violence and sexual assaults.

Forensic science has made a lot of progress in the medical field, but still ocular signs are not so well known to forensic scientists, whereas ocular signs play a very important role in forensic science. The major goal of this study to describe the importance of eye related clues in forensic science to solve the crime scenes and know the actual cause of death for the conviction of the criminals and save an innocent persons. Narrative review was done to review the articles available on the PubMed, Google Scholar, Research Gate, Web of Science and Medline related to the forensic optometry about the various methods of identification and strategies to deal with the forensic cases. The review covered studies that focused on interventions for forensic optometry and eye-related clues to the current study target. Twenty-seven of the 54 studies that met the inclusion criteria yielded good results, and all examined the significance of eye-related clues in forensic optometry. Peer reviewed articles/studies were referred to ascertain the eye related clues in the forensic science. Some authors suggest that eye related clues are very important aspects towards the forensic science and done the clinical research on that aspects. Criminal cases, domestic abuse and sexual assault affect people of all socioeconomic backgrounds. As a result, these traits would be highly valuable in determining the way of death and the chronological history of events leading up to a crime.

Approaching headaches and facial pains in eye care practice.

Headaches and facial pains are among the most frequent ailments seen in outpatient or emergency settings. Given the fact that some of the primary headaches and facial pains mimic the characteristic patterns seen in ocular diseases and related conditions, it is fairly common for these situations to be sent to an ophthalmology or optometry clinic and misdiagnosed as ocular headaches. This may result in a delay in starting an appropriate therapy, therefore extending the patient's illness. This review article aims to help the practitioners in understanding common causes of headaches and facial pains, approaching such cases in eye OPD and differentiating them for similar ocular conditions to impart an appropriate treatment or referral.

Deep Inception-ResNet: A Novel Approach for Personalized Prediction of Cumulative Pregnancy Outcomes in Vitro Fertilization Treatment (IVF).

Background: Infertility is one of the major causes of socioeconomic stress worldwide due to social stigma and stressful lifestyles. Despite technological advances, couples still undergo several IVF cycles for conceiving without knowing their true prognosis which is causing a huge social and medical impact, and the live birth rate continues to be relatively low (~ 25%). A prediction model that predicts IVF prognosis accurately considering the pre-treatment parameters before starting the IVF cycle will help clinicians and patients to make better-informed choices. Methods: In this study, clinical details of 2268 patients with 79 features who underwent IVF/ICSI procedure from January 2018 to December 2020, at the Center of IVF and Human Reproduction, Sir Ganga Ram Hospital were retrospectively collected. The machine learning model was developed considering features such as maternal age, number of IVF cycle, type of infertility, duration of infertility, AMH, indication for IVF, sperm type, BMI, embryo transfer, and ß-hCG value at the end of a fresh cycle and/or one subsequent frozen embryo transfer cycle was selected as the measure of outcome. Results: Compared to other classifiers, for an 80:20 train-test split with feature selection, the proposed Deep Inception-Residual Network architecture-based neural network gave the best accuracy (76%) and ROC-AUC score of 0.80. For tabular datasets, the applied approach has remained unexplored in previously made studies for reproductive health. Conclusion: This model is the starting point for providing a personalized prediction of a successful outcome for an infertile couple before they enter the IVF procedure. Supplementary Information: The online version contains supplementary material available at 10.1007/s13224-023-01773-9.

Hand Foot Mouth Disease During the SARS-CoV-2 Pandemic: A Multicentric Study.

OBJECTIVES: This prospective observational study aimed to identify the current trend of the circulating viral strains responsible for HFMD outbreak in four tertiary care centers in Rajasthan, amidst the COVID-19 pandemic (April-October 2022). METHODS: Cases with suspected HFMD, presenting to our skin outpatient department were assessed clinically and serologically (IgM antibodies against coxsackie virus (CV) A6, A16 and enterovirus 71) for evidence of the disease. RESULTS: We identified 718 new HFMD patients (161 adults) with peaks in May and August, 2022. Male:female ratio decreased with increasing age. Most children were asymptomatic. A total of 385/409 patients assessed serologically, were found positive, most commonly against CV-A6. CONCLUSION: Though HFMD typically affects young children, an unusually higher proportion of adults were affected during the current pandemic. There were some subtle differences between pediatric and adult presentation of HFMD.

Co-occurrence of mycotoxins: A review on bioanalytical methods for simultaneous analysis in human biological samples, mixture toxicity and risk assessment strategies.

Mycotoxins are the toxic chemical substances that are produced by various fungal species and some of these are harmful to humans. Mycotoxins are ubiquitous in nature and humans could be exposed to multiple mycotoxins simultaneously. Unfortunately, exposure to mixed mycotoxins is not very well studied. Various studies have demonstrated the capacity of mycotoxins to show synergistic effect in the presence of other mycotoxins, thus, increasing the risk of toxicity. Hence, it is important to monitor mixed mycotoxins in human biological samples which would serve as a crucial information for risk assessment. Through this review paper, we aim to summarize the mixture toxicity of mycotoxins and the various bio-analytical techniques that are being used for the simultaneous analysis of mixed mycotoxins in human biological samples. Different sample preparation and clean-up techniques employed till date for eliminating the interferences from human biological samples without affecting the analyses of the mycotoxins are also discussed. Further, a brief introduction of risk assessment strategies that have been or could be adopted for multiple mycotoxin risk assessments is also mentioned. To the best of our knowledge, this is the first review that focuses solely on the occurrence of multiple mycotoxins in human biological samples as well as their risk assessment strategies.

Candle soot derived carbon nanoparticles: An assessment of cellular and progressive toxicity using Drosophila melanogaster model.

The biomedical demand of the nanomaterials is continuously increasing due to their wide range of applications in the field. However, before the implementation of these nanomaterials, toxicity assessment is essential for its safe usage. In the present study, the toxicity of carbon nanoparticles (CNPs) was investigated which was derived from candle soot and compared with commercially available multi-walled carbon nanotubes (CNTs) by using Drosophila melanogaster as a model system. First instar Drosophila larvae were exposed to CNPs as well as CNTs, and the toxic effects of these nanomaterials were compared. The result shows that both nanomaterials enhance the level of reactive oxygen species and oxidative stress in the Drosophila, which leads to the upregulation of heat shock proteins that may cause cytotoxicity in exposed Drosophila larvae. In contrast, exposure to CNPs and CNTs did not affect the developmental period of the larvae. Morphology of the internal organs, brain, gut and Malpighian tubules was also not altered in the exposed larvae. Similarly, no change observed in the cytoskeleton (F-actin) of these organs. Reproductive performance was slightly reduced in the case of CNPs compare to control. However, CNTs exposure did not show any significant effect on the reproductive performance of the flies that emerged from exposed larvae in comparison to control. Hence the study concludes that exposure to CNPs and CNTs cause a moderate level of cytotoxicity in Drosophila. The study also indicates that the inexpensive CNPs may use as an alternative to expensive CNTs for biomedical and biological applications.

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.

Endophytes of Withania somnifera modulate in planta content and the site of withanolide biosynthesis.

Tissue specific biosynthesis of secondary metabolites is a distinguished feature of medicinal plants. Withania somnifera, source of pharmaceutically important withanolides biosynthesizes withaferin-A in leaves and withanolide-A in roots. To increase the in planta withanolides production, a sustainable approach needs to be explored. Here, we isolated endophytes from different parts of W. somnifera plants and their promising role in in planta withanolide biosynthesis was established in both in-vivo grown as well in in-vitro raised composite W. somnifera plants. Overall, the fungal endophytes improved photosynthesis, plant growth and biomass, and the root-associated bacterial endophytes enhanced the withanolide content in both in-vivo and in-vitro grown plants by modulating the expression of withanolide biosynthesis genes in leaves and roots. Surprisingly, a few indole-3-acetic acid (IAA)-producing and nitrogen-fixing root-associated endophytes could induce the biosynthesis of withaferin-A in roots by inducing in planta IAA-production and upregulating the expression of withanolide biosynthesis genes especially MEP-pathway genes (DXS and DXR) in roots as well. Results indicate the role of endophytes in modulating the synthesis and site of withanolides production and the selected endophytes can be used for enhancing the in planta withanolide production and enriching roots with pharmaceutically important withaferin-A which is generally absent in roots.

Production of anti-cancer triterpene (betulinic acid) from callus cultures of different Ocimum species and its elicitation.

Betulinic acid (BA), a pentacyclic triterpenoid, is gaining unmatched attention owing to its unique anti-cancer activity with selective melanoma growth inhibition without damaging normal cells. It is also well-known for its multifaceted pharmacokinetics, entailing antibacterial, antimalarial, anti-HIV and antioxidant merits. Considering the escalating demand with diminishing bioresource of this molecule, the present study was undertaken that revealed the untapped potentials of Ocimum calli, contrasting to that in the in vitro derived leaves, as effective production alternative of BA in three out of four tested species (i.e. Ocimum basilicum, Ocimum kilimandscharicum, Ocimum sanctum excluding Ocimum grattisimum). Callus inductions were obtained in all the four species with different 2,4-dichlorophenoxyacetic acid (2,4-D)/α-naphthaleneacetic acid (NAA) concentrations with kinetin. Notably, 2,4-D favoured maximum callus growth in all whereas NAA proved beneficial for the highest metabolite yield in the calli of each BA-producing species. The O. basilicum calli demonstrated the maximum growth (growth index (GI) 678.7 ± 24.47) and BA yield (2.59 ± 0.55 % dry weight [DW]), whereas those in O. kilimandscharicum (GI 533.33 ± 15.87; BA 1.87 ± 0.6 % DW) and O. sanctum (GI 448 ± 16.07; BA 0.39 ± 0.12 % DW) followed a descending order. The O. gratissimum calli revealed minimum growth (GI 159 ± 13.25) with no BA accumulation. Elicitation with methyl jasmonate at 200-µM concentration after 48-h exposure doubled the BA yield (5.10 ± 0.18 % DW) in NAA-grown O. basilicum calli compared to that in the untreated counterpart (2.61 ± 0.19 % DW), which further enthused its future application.