Assessing the Impact of the Portfolio Workshop and the Use of the Rubric Matrix for Reflective Writing on Medical Faculty and Students.

Purpose The portfolio can be used as a crucial tool for self-reflection, which allows us not only to showcase achievements but also course correct on our personal and professional journey. However, there is a significant lack of awareness among medical professionals about portfolios. Arranging a workshop to impart this knowledge could be a potential mitigation approach. This study aims to assess the impact of workshops on portfolios on students' and faculty's knowledge. In addition, the study also analyzes the effect of using rubrics on reflective writing skills. Method A portfolio workshop was organized for the medical faculty and students in the Bundelkhand Government Medical College, Sagar, M.P. The Kirkpatrick model of training evaluation along with a rubric for the evaluation of reflective writing skills were used to measure the effectiveness of the workshop. Pre and post-tests for the workshop, pre and post-reflective writing skills, and workshop feedback were collected using questionnaires. The Shapiro-Wilk test and the Wilcoxon signed rank test were applied to the data collected. Results Out of 89 registrations for the workshop, only 81 people consented to the workshop and participated in the study. The total number of faculty was only 17 and the rest were students from all the phases. Both the Shapiro-Wilk test and the Wilcoxon signed rank test showed a significantly small p-value, stating that there was a significant positive impact on the knowledge, perception, and effectiveness of the workshop. Conclusion This study clearly outlines the positive impact of conducting a workshop on portfolios. A significant increase in participants' knowledge of portfolios is identified. Similarly, employing rubrics has a significant increase in the quality of reflective writing skills.

Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for 'programmable addition via site-specific targeting elements', a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.

Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Histo-pathological patterns in hysterectomy specimens at a tertiary care centre in India.

It is of interest to document histo-pathological patterns in hysterectomy specimens at tertiary care centre in India. This study included 442 cases. In this study, leiomyoma (9.17 %) was the most common preoperatively clinical diagnosis made in hysterectomy specimen. In this study, uterine fibroid showed a 90.47% correlation between pre-operative and histological findings. There was a 50 % correlation noted between adenomyosis and endocervical polyp.

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

The Role of Antioxidant Agent (N-Acetylcysteine) in Oleic Acid-Induced Acute Lung Injury in a Rat Model.

Context Reactive oxygen species (ROS) produced by inflammatory cells play a major role in mediating lung injury in sepsis or hyperoxic lung injury. Aims N-Acetylcysteine (NAC), an antioxidant, was examined in this research to see whether it helps prevent acute lung injury (ALI). Materials and methods Experiments were performed on Charles-Foster strain healthy male adult albino rats. All the animals were randomly divided into one control and two experimental groups. In control/group I, saline was administered, and cardiorespiratory parameters were recorded. Oleic acid (OA) was administered in group II to produce ALI. In group III, OA was administered to NAC-pretreated rats, and cardiorespiratory parameters were recorded to observe the effect of NAC on ALI. This study used analysis of variance (ANOVA) with two factors and a post hoc test (multiple comparisons - least significant difference (LSD) test) for statical analysis. For determining survival time, the Mantel-Cox test and Kaplan-Meier survival curves were used. A P value < 0.05 was considered significant. Results Respiratory arrest, pulmonary edema, and reduced partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) ratio were all indications of OA-induced ALI in rats. The animals in the NAC + OA group had better respiratory and cardiac statistics than those in the OA alone group, and their survival duration was extended. However, NAC pretreatment could not protect the animals against the development of pulmonary edema. Conclusions These observations indicate that NAC (an antioxidant agent) protected rats against ALI in the initial phase and prolonged the survival time but failed to prevent the development of pulmonary edema.

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ.

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

Response Surface Methodology-Genetic Algorithm Based Medium Optimization, Purification, and Characterization of Cholesterol Oxidase from Streptomyces rimosus.

The applicability of the statistical tools coupled with artificial intelligence techniques was tested to optimize the critical medium components for the production of extracellular cholesterol oxidase (COD; an enzyme of commercial interest) from Streptomyces rimosus MTCC 10792. The initial medium component screening was performed using Placket-Burman design with yeast extract, dextrose, starch and ammonium carbonate as significant factors. Response surface methodology (RSM) was attempted to develop a statistical model with a significant coefficient of determination (R2 = 0.89847), followed by model optimization using Genetic Algorithm (GA). RSM-GA based optimization approach predicted that the combination of yeast extract, dextrose, starch and ammonium carbonate at concentrations 0.99, 0.8, 0.1, and 0.05 g/100 ml respectively, has resulted in 3.6 folds increase in COD production (5.41 U/ml) in comparison with the un-optimized medium (1.5 U/ml). COD was purified 10.34 folds having specific activity of 12.37 U/mg with molecular mass of 54 kDa. The enzyme was stable at pH 7.0 and 40 °C temperature. The apparent Michaelis constant (Km) and Vmax values of COD were 0.043 mM and 2.21 µmol/min/mg, respectively. This is the first communication reporting RSM-GA based medium optimization, purification and characterization of COD by S. rimosus isolated from the forest soil of eastern India.

Leishmania donovani molecules recognized by sera of filaria infected host facilitate filarial infection.

We earlier found that F6 fraction of human filaria Brugia malayi cross-reacted with sera of Leishmania donovani infected hamsters and immunization with F6 inhibited both filarial and leishmanial infections. In the present study, we identified a 52.9-93.6 kDa fraction (Ld1) of L. donovani that cross-reacted with sera of B. malayi infected animals and investigated effect of Ld1 on filarial infection. Immunization of BALB/c mice with Ld1 facilitated B. malayi infection with remarkable increase in parasite burden. Facilitation of filarial infection was associated with downregulated cell proliferation, IL-5, IL-13, IFN-γ, TNF-α, and IL-2 levels and upregulated IL-4 and TGF-ß. Ld1 exposure also suppressed MHC class-I, MHC class-II, and FcεR1 expression, and phagocytosis in naive mouse macrophages, and CD4+, CD8+, and CD19+ cell population in mouse spleen. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight-mass spectrometry revealed eight proteins in Ld1: putative heat shock protein (HSP) 70-related protein 1, HSP70 mitochondrial precursor, alanine aminotransferase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, protein disulfide isomerase, putative ATPase beta subunit, trypanothione reductase, and a hypothetical protein. HSP70 protein mitochondrial precursor and trypanothione reductase showed homology with Trypanosoma cruzi and L. donovani, respectively, and the rest 6 proteins including hypothetical protein bear homology with L. infantum. In conclusion, the present study for the first time shows that immunization with filarial cross-reactive Ld1 fraction of L. donovani facilitates filarial infection by modulating Th1 and Th2 responses. Ld1 molecules may therefore facilitate filarial infection in filaria-leishmania co-infection.

Identification of 1-[4-Benzyloxyphenyl)-but-3-enyl]-1H-azoles as New Class of Antitubercular and Antimicrobial Agents.

A series of 1-[(4-benzyloxyphenyl)-but-3-enyl]-1H-azoles has been identified as potent antitubercular agents against Mycobacterium tuberculosis. Synthesis of compounds involved acid catalyzed ring-opening of cyclopropyl ring of phenyl cyclopropyl methanols followed by nucleophilic attack of the azoles on the carbocation intermediates. Several of the compounds 26, 34, and 36 exhibited significant antitubercular activities with MIC value as low as 1.56, 1.56, and 0.61 µg/mL, respectively, comparable to many standard drugs. These compounds were also screened against other strains of bacteria and fungi, and few of them showed good antifungal activity against A. fumigatus, responsible for lung infection.