Engineering Artificial Small RNAs for Conditional Gene Silencing in <i>Escherichia coli</i>

It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the <i>trans</i>-acting sRNAs in bacteria interact with the 5′ untranslated region (UTR) and/or the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and/or mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation. In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired endogenous genes in <i>Escherichia coli</i>. Using a fluorescent reporter gene that was translationally fused to a native 5′ mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a randomized antisense domain. Artificial sRNAs that posttranscriptionally repress two endogenous genes <i>ompF</i> and <i>fliC</i> were isolated and characterized. We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology

AI-assisted Single-Image Full-Frame Camera Calibration for Space-Constrained Stereoscopic Systems

Camera calibration plays a fundamental role in the wake of the newly emerging image data-driven technologies, where pinpoint accuracy in data is vital to the successful functioning of these systems. Conventional camera calibration algorithms require manual object placement, a process that can be exceptionally time-consuming and labor-intensive, particularly in scenarios where space constraints or delicate equipment are involved. We present an innovative calibration object and AI-aided pre-calibration routine with a specific emphasis on space-restricted environments. The proposed methodology obviates the need for manual multi-image acquisition. This is achieved by fabricating the novel calibration object, which contains 20 checkerboards in different positions and orientations. The precursor routine, assisted by an AI model, isolates and processes the individual checkerboards, which is then used as input for the camera calibration. We report an accuracy of 99.92% for ML-assisted checkerboard separation, with procedure time improved by nearly 64x and overall corrected reprojection error consistently below 0.5 pixels. Incorporating the proposed calibration routine into a 3D vascular imaging stereovision system, we demonstrate a depth resolution of 0.5mm

Lipovelutibols A–D: Cytotoxic Lipopeptaibols from the Himalayan Cold Habitat Fungus <i>Trichoderma velutinum</i>

Four novel lipovelutibols A (<b>1</b>), B (<b>2</b>), C (<b>3</b>), and D (<b>4</b>) containing six amino acid residues with leucinol at the C-terminus and a fatty acyl moiety (<i>n</i>-octanoyl) at its N-terminus were isolated from the psychrotrophic fungus <i>Trichoderma velutinum</i> collected from the Himalayan cold habitat. The structures (<b>1</b>–<b>4</b>) were determined by NMR and MS/MS, and the stereochemistry of amino acids by Marfey’s method. Lipopeptaibols <b>2</b> and <b>4</b> were found to contain d-isovaline, a nonproteinogenic amino acid, but lacked α-aminoisobutyric acid, characteristic of peptaibols. Cytotoxic activity of <b>2</b> and <b>4</b> was observed against HL-60, LS180, MDA-MB-231, and A549 cancer cell lines

Supplementary_Table_1 - Effects of Turmeric and Curcumin Dietary Supplementation on Human Gut Microbiota: A Double-Blind, Randomized, Placebo-Controlled Pilot Study

<p>Supplementary_Table_1 for Effects of Turmeric and Curcumin Dietary Supplementation on Human Gut Microbiota: A Double-Blind, Randomized, Placebo-Controlled Pilot Study by Christine T. Peterson, Alexandra R. Vaughn, Vandana Sharma, Deepak Chopra, Paul J. Mills, Scott N. Peterson, and Raja K. Sivamani in Journal of Evidence-Based Integrative Medicine</p

Design, Synthesis, ADME, and Anticancer Studies of Newer <i>N</i>‑Aryl-5-(3,4,5-Trifluorophenyl)-1,3,4-Oxadiazol-2-Amines: An Insight into Experimental and Theoretical Investigations

In continuance of our investigation into the anticancer activity of oxadiazoles, we report here the preparation of 10 new 1,3,4-oxadiazole analogues using the scaffold hopping technique. We have prepared the oxadiazoles having a common pharmacophoric structure (oxadiazole linked aryl nucleus) as seen in the reported anticancer agents IMC-038525 (tubulin inhibitor), IMC-094332 (tubulin inhibitor), and FATB (isosteric replacement of the S of thiadiazole with the O of oxadiazole). All of the oxadiazole analogues were predicted for their absorption, distribution, metabolism, and excretion (ADME) profiles and toxicity studies. All of the compounds were found to follow Lipinski’s rule of 5 with a safe toxicity profile (Class IV compound) against immunotoxicity, mutagenicity, and toxicity. All of the compounds were synthesized and characterized using spectral data, followed by their anticancer activity tested in a single-dose assay at 10 μM as reported by the National Cancer Institute (NCI US) Protocol against nearly 59 cancer cell lines obtained from nine panels, including non-small-cell lung, ovarian, breast, central nervous system (CNS), colon, leukemia, prostate, and cancer melanoma. N-(2,4-Dimethylphenyl)-5-(3,4,5-trifluorophenyl)-1,3,4-oxadiazol-2-amine (6h) displayed significant anticancer activity against SNB-19, OVCAR-8, and NCI-H40 with percent growth inhibitions (PGIs) of 86.61, 85.26, and 75.99 and moderate anticancer activity against HOP-92, SNB-75, ACHN, NCI/ADR-RES, 786-O, A549/ATCC, HCT-116, MDA-MB-231, and SF-295 with PGIs of 67.55, 65.46, 59.09, 59.02, 57.88, 56.88, 56.53, 56.4, and 51.88, respectively. The compound 6h also registered better anticancer activity than Imatinib against CNS, ovarian, renal, breast, prostate, and melanoma cancers with average PGIs of 56.18, 40.41, 36.36, 27.61, 22.61, and 10.33, respectively. Molecular docking against tubulin, one of the appealing cancer targets, demonstrated an efficient binding within the binding site of combretastatin A4. The ligand 6h (docking score = −8.144 kcal/mol) interacted π-cationically with the residue Lys352 (with the oxadiazole ring). Furthermore, molecular dynamic (MD) simulation studies in complex with the tubulin-combretastatin A4 protein and ligand 6h were performed to examine the dynamic stability and conformational behavior

Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor

<div><p>In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, <i>Drosophila melanogaster</i>. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.</p></div

Western blots showing the levels of EcR and USP in knockdown males compared to control males.

<p>The EcR panel represents EcR levels in accessory glands from EcR control (+ lane, EcR), EcR knockdown (-lane, EcR), USP control (+ lane, USP) and USP knockdown (- lane, USP). Similarly, the USP panel represents the USP levels observed in accessory glands from above groups. Blots probed with α-tubulin antibodies (α-tubulin panels) served as controls for protein loading. Knockdowns were specific to the targeted hormone receptor. Further, the deficiency of EcR did not affect USP levels and vice-versa.</p

Reproductive performance of females mated to EcR or USP knockdown males over period of 10 days.

<p>Panel A represents the overall fecundity (total no. of eggs laid/10 days) of mated females while Panels B and C represent day wise fecundity (no. of egg laid/day from day1-10) of mated females. The overall fecundity of EcR knockdown mates was significantly lower compared to controls (***<i>p</i><0.0001). However, overall fecundity of USP knockdown mates was comparable to their controls. (<i>p</i> = 0.08). Further, EcR knockdown mates laid eggs for 24hr ASM, albeit at significantly fewer numbers (***<i>p</i><0.0001) when compared to control and did not lay eggs from days 2–10. USP knockdown mates did not deviate from controls on day wise egg laying. Interestingly, there were no progeny from the eggs laid by EcR knockdown mates over period of 10 days (overall fertility, ***<i>p<</i>0.0001, Panel D; day-wise fertility, ***<i>p<</i>0.0001 Panel E). USP knockdown or control mates had comparable overall (Panel D; <i>p =</i> 0.3, USP) as well as day wise fertility (Panel F; <i>p =</i> 0.06 lowest) EcR control and knockdown mates differ significantly on total % hatchability (Panel G; EcR, ***<i>p</i><0.0001) as well as % hatchability on day 1 (Panel H; ***<i>p<</i>0.0001). USP control and knockdown mates had significant differences in % hatchability on days 8 and 10 (Panel I; <i>p</i> = 0.01) but that did not have a significant bearing on the overall % hatchability (Panel H; <i>p</i> = 0.3). Values given here are Mean±SEM involving at least 15–30 females depending on the hormone receptor.</p

The effect of knockdown of different hormone receptors on the fertility of mated females.

<p>To identify the nuclear/hormone receptor involved in male fertility, 19 receptors were knocked down, individually, in accessory gland specific manner in the male reproductive tract and were allowed to mate with virgin females. Shown here is the number [Mean±Standard Error (SEM)] of progeny produced by females mated to knockdown or control males over a period of 10 days. Mates of <i>met</i> (Methoprene tolerant, a juvenile hormone receptor, **<i>p<</i>0.001) knockdown males produced significantly fewer progeny when compared to their controls and also those in strain background control (Jhe). Interestingly, females mated to EcR knockdown males failed to produce progeny (EcR; ***<i>p<</i>0.0001; Bonferroni corrected p value for significance is <i>p<</i>0.002). However, fertility of females mated to USP (<i>p =</i> 0.27) or the remaining 16 hormone receptor knockdown males was not significantly different from their respective controls. Number of females ranged from 15–45 depending on the hormone receptor analyzed.</p

The effect of knockdown of EcR or USP on the structure of male accessory glands.

<p>To assess the effect of depletion of EcR or USP, accessory glands were analyzed either at the ultrastructural level (panels at the top). Depicted at the top are the electron micrographs of male accessory glands from (A) EcR control (B) EcR knockdown (C) USP control and (D) knockdown males. Accessory glands from EcR control, USP control and USP knockdown males show normal protein filamentous structures (labeled as f) throughout in their lumen. However, glands of EcR knockdown have extreme vacuolization (v) and lack filamentous structures in the lumen. A minimum of five tissues from each group was used for ultrastructural analysis.</p