Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Functions of the Bloom Syndrome Helicase N-terminal Intrinsically Disordered Region.

Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human BLM cause the autosomal recessive disorder Bloom Syndrome, characterized by predisposition to numerous types of cancer. Prior studies of Drosophila Blm mutants lacking helicase activity or protein have shown sensitivity to DNA damaging agents, defects in repairing DNA double-strand breaks (DSBs), female sterility, and improper segregation of chromosomes in meiosis. Blm orthologs have a well conserved and highly structured RecQ helicase domain, but more than half of the protein, particularly in the N-terminus, is predicted to be unstructured. Because this region is poorly conserved across multicellular organisms, we compared closely related species to identify regions of conservation, potentially indicating important functions. We deleted two of these Drosophila-conserved regions in D. melanogaster using CRISPR/Cas9 gene editing and assessed the effects on different Blm functions. Each deletion had distinct effects on different Blm activities. Deletion of either conserved region 1 (CR1) or conserved region 2 (CR2) compromised DSB repair through synthesis-dependent strand annealing and resulted in increased mitotic crossovers. In contrast, CR2 is critical for embryonic development but CR1 is not as important. CR1 deletion allows for proficient meiotic chromosome segregation but does lead to defects in meiotic crossover designation and patterning. Finally, deletion of CR2 does not lead to significant meiotic defects, indicating that while each region has overlapping functions, there are discreet roles facilitated by each. These results provide novel insights into functions of the N-terminal disordered region of Blm.

The analgesic effect of transmuscular quadratus lumborum block versus erector spinae plane block for women undergoing elective Caesarean section: A randomized controlled trial.

Background: We aimed to compare the analgesic effect and quality of recovery of transmuscular quadratus lumborum block (QLB) and erector spinae plane block (ESPB) in the presence of multimodal analgesia following Caesarean section. Methods: This randomised controlled trial included full-term pregnant women who underwent elective Caesarean section under spinal anaesthesia without intrathecal morphine. The included women were randomised to receive either bilateral QLB (n = 51) or bilateral thoracic ESPB (n = 51), in addition to the control group (n = 51) who did not receive any block. All participants received regular paracetamol (1 g/6h) and diclofenac (50 mg/8 h). Intravenous morphine was used as a rescue analgesic. The primary outcome was time to first morphine requirement. Secondary outcomes included total postoperative morphine consumption and total Obstetric Quality of Recovery-11 (ObsQoR-11) score. Results: We analysed 51, 50 and 48 patients in the QLB, ESPB and control groups, respectively. The time to first morphine requirement in both QLB and ESPB groups was longer than that in the control group (median [quartiles] time: 6 [6,12] h, 6 [6,6] h and 4 [3,4] h, respectively; p-value <0.001), without significant differences between the two former groups. The total morphine consumption in both QLB and ESPB groups was lower than that in the control group (median [quartiles]:0 [0,5] mg, 0 [0,5] mg and 25 [25,30] mg, respectively; p-value <0.001), without significant differences between the two former groups. The QLB and ESPB groups had comparable ObsQoR-11 score, and both groups' scores were higher than the control group. Conclusion: In patients undergoing elective Caesarean section under spinal anaesthesia without intrathecal morphine, both QLB and ESPB provided superior analgesia and quality of recovery compared to the standard care, without significant difference between the two blocks.

Divergent directed evolution of a TetR-type repressor towards aromatic molecules.

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.

Adjunctive proximal aortic and aortodistal prosthetic wrapping of vascular anastomoses for Adamantiadis-Behçet's aortic/aortoiliac aneurysms: A revisit.

BACKGROUND: We described our local experiences with a single-layer wrapping technique for the vascular anastomoses in patients with Adamantiadis-Behçet's aortic/aortoiliac aneurysms using InterGard Silver-impregnated Dacron® patch prosthesis. METHODS: Between January 2013 to December 2019, we retrospectively reviewed 20 patients presented with Adamantiadis-Behçet's aortic/aortoiliac aneurysms. All patients presented with Adamantiadis-Behçet's aortic/aortoiliac aneurysms. Two groups were analyzed, Group I, considered as a control group (n = 20). While group II (n = 20), of which prosthetic wrapping was performed. Follow up took place for a maximum of 24 months. RESULTS: during a six-year retrospective study period, 20 patients were recruited. They included 15 males and five females (ratio 3:1). The median age was 30.5 ± 4.2 years. Anastomotic pseudoaneurysms were reported in group I (control, [non-wrapping group]). While group II doesn't (wrapping group). Paired samples t test revealed a significant difference between those underwent wrapping and those with non-wrapping (p = .019 and .038). False aneurysms were reported in 80% of the non-wrapping group as estimated by the Kaplan-Meier curves. While Log-rank test results revealed a significant difference between both the studied groups (p < .008). Primary graft patency was 90% at 24 months as reported by the Kaplan-Meier survival method. CONCLUSIONS: adjunctive wrapping for vascular anastomoses using Intergard Silver-impregnated Dacron® patch in patients with Adamantiadis-Behçet's aortic/aortoiliac aneurysms is an applicable, simple, and reliable technique. It was associated with low morbidity and mortality rates. Moreover, we discussed a relatively old technique aiming to explore its success and safety in treating arterial aneurysms in Adamantiadis-Behçet's disease patients.

A Synthetic Biosensor for Detecting Putrescine in Beef Samples.

Biogenic amines (BAs) are toxicological risks present in many food products. Putrescine is the most common foodborne BA and is frequently used as a quality control marker. Currently, there is a lack of regulation concerning safe putrescine limits in food as well as outdated food handling practices leading to unnecessary putrescine intake. Conventional methods used to evaluate BAs in food are generally time-consuming and resource-heavy with few options for on-site analysis. In response to this challenge, we have developed a transcription factor-based biosensor for the quantification of putrescine in beef samples. In this work, we use a naturally occurring putrescine responsive repressor-operator pair (PuuR-puuO) native to Escherichia coli. Moreover, we demonstrate the use of the cell-free putrescine biosensor on a paper-based device that enables rapid low-cost detection of putrescine in beef samples stored at different temperatures. The results presented demonstrate the potential role of using paper-based biosensors for on-site testing, particularly as an index for determining meat product stability and quality.

Regulation of mitochondrial temperature in health and disease.

Mitochondrial temperature is produced by various metabolic processes inside the mitochondria, particularly oxidative phosphorylation. It was recently reported that mitochondria could normally operate at high temperatures that can reach 50℃. The aim of this review is to identify mitochondrial temperature differences between normal cells and cancer cells. Herein, we discussed the different types of mitochondrial thermosensors and their advantages and disadvantages. We reviewed the studies assessing the mitochondrial temperature in cancer cells and normal cells. We shed the light on the factors involved in maintaining the mitochondrial temperature of normal cells compared to cancer cells.

A Versatile Transcription Factor Biosensor System Responsive to Multiple Aromatic and Indole Inducers.

Allosteric transcription factor (aTF) biosensors are valuable tools for engineering microbes toward a multitude of applications in metabolic engineering, biotechnology, and synthetic biology. One of the challenges toward constructing functional and diverse biosensors in engineered microbes is the limited toolbox of identified and characterized aTFs. To overcome this, extensive bioprospecting of aTFs from sequencing databases, as well as aTF ligand-specificity engineering are essential in order to realize their full potential as biosensors for novel applications. In this work, using the TetR-family repressor CmeR from Campylobacter jejuni, we construct aTF genetic circuits that function as salicylate biosensors in the model organisms Escherichia coli and Saccharomyces cerevisiae. In addition to salicylate, we demonstrate the responsiveness of CmeR-regulated promoters to multiple aromatic and indole inducers. This relaxed ligand specificity of CmeR makes it a useful tool for detecting molecules in many metabolic engineering applications, as well as a good target for directed evolution to engineer proteins that are able to detect new and diverse chemistries.

BCG vaccination and the risk of COVID 19: A possible correlation.

Bacillus Calmette-Guérin (BCG) vaccine is currently used to prevent tuberculosis infection. The vaccine was found to enhance resistance to certain types of infection including positive sense RNA viruses. The current COVID-19 pandemic is caused by positive sense RNA, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A higher mortality rate of COVID-19 patients was reported in countries where BCG vaccination is not routinely administered, when compared to the vaccinated ones. We hypothesized that BCG vaccine may control SARS-CoV2 infection via modulating the monocyte immune response. We analyzed GSE104149 dataset to investigate whether human monocytes of BCG-vaccinated individuals acquire resistance to SARS-CoV-2 infection. Differentially expressed genes obtained from the dataset were used to determine enriched pathways, biological processes, and molecular functions for monocytes post BCG vaccination. Our data show that BCG vaccine promotes a more effective immune response of monocytes against SARS-CoV2, but probably not sufficient to prevent the infection.

Hepatocellular carcinoma cell line-microenvironment induced cancer-associated phenotype, genotype and functionality in mesenchymal stem cells.

Mesenchymal stromal cells (MSCs) have shown promise in liver cancer treatment. However, when MSCs are recruited to hepatic site of injury, they acquire cancerous promoting phenotype. AIMS: To assess the influence of Hepatocellular carcinoma (HCC) microenvironment on human adipose MSCs (hA-MSCs) and predict hA-MSCs intracellular miRNAs role. MATERIALS AND METHODS: After indirect co-culturing with Huh-7 cells, hA-MSCs were characterized via cell cycle profile, proliferation and migration potentials by MTT and scratch assays respectively. Functional enrichment analysis of deregulated proteins and miRNA targets was also analyzed. KEY FINDINGS: Co-cultured hA-MSCs could acquire a cancer-associated phenotype as shown by upregulation of CAF, cancer markers, and downregulation of differentiation markers. Migration of these cancer-associated cells was increased concomitantly with upregulation of adhesion molecules, but not epithelial to mesenchymal transition markers. Co-cultured cells showed increased proliferation confirmed by downregulation in cell percentage in G0/G1, G2/M and upregulation in S phases of cell cycle. Upregulation of miR-17-5p and 615-5p in co-cultured hA-MSCs was also observed. Functional enrichment analysis of dysregulated proteins in co-cultured hA-MSCs, including our selected miRNAs targets, showed their involvement in development of cancer-associated characteristics. SIGNIFICANCE: This study suggests an interaction between tumor cells and surrounding stromal components to generate cancer associated phenotype of some CAF-like characteristics, known to favor cancer progression. This sheds the light on the use of hA-MSCs in HCC therapy. hA-MSCs modulation may be partially achieved via dysregulation of intracellular miR17-5P and 615-5p expression, suggesting an important role for miRNAs in HCC pathogenesis, and as a possible therapeutic candidate.

Incidental Unruptured Isolated Left Common Iliac Artery Aneurysm: A Rare Entity.

Iliac artery aneurysms (IAA) are a rare entity. The etiology behind IAA is unclear; however, it is typically degenerative or atherosclerotic in origin. In patients presenting with sudden rupture requiring emergent surgery, mortality rates are high, signifying the need for prompt diagnosis and treatment. We report a case of an incidentally found unruptured isolated left common iliac artery aneurysm in an 80-year-old man. Management with aortofemoral angioplasty was successfully performed for this patient.

Dysregulated MicroRNA Fingerprints and Methylation Patterns in Hepatocellular Carcinoma, Cancer Stem Cells, and Mesenchymal Stem Cells.

Hepatocellular carcinoma (HCC) is one of the top causes of cancer mortality worldwide. Although HCC has been researched extensively, there is still a need for novel and effective therapeutic interventions. There is substantial evidence that initiation of carcinogenesis in liver cirrhosis, a leading cause of HCC, is mediated by cancer stem cells (CSCs). CSCs were also shown to be responsible for relapse and chemoresistance in several cancers, including HCC. MicroRNAs (miRNAs) constitute important epigenetic markers that regulate carcinogenesis by acting post-transcriptionally on mRNAs, contributing to the progression of HCC. We have previously shown that co-culture of cancer cells with mesenchymal stem cells (MSCs) could induce the reprogramming of MSCs into CSC-like cells. In this review, we evaluate the available data concerning the epigenetic regulation of miRNAs through methylation and the possible role of this regulation in stem cell and somatic reprogramming in HCC.

Ultrasound-guided quadratus lumborum block for postoperative pain control in patients undergoing unilateral inguinal hernia repair, a comparative study between two approaches.

BACKGROUND: Early postoperative ambulation and reduction of hospital stay necessitate efficient postoperative analgesia. Quadrates Lumborum Block (QLB) has been described to provide adequate postoperative analgesia after abdominal surgery. This randomized comparative trial was designed to compare the duration of analgesia provided by two different QLB approaches; the posterior QLB (QLB-2) and transmuscular QLB (QLB-3) in patients undergoing surgical repair of unilateral inguinal hernia. METHODS: Forty patients, aged from 18 to 50 years, ASA physical status I or II, scheduled for unilateral inguinal hernia repair were enrolled. At the end of the surgical procedure and before recovery from general anesthesia, Patients were randomly assigned into two groups to receive either posterior QLB (Group QLB-2) or transmuscular QLB (Group QLB-3) using 20 ml 0.25% bupivacaine. Duration of analgesia, postoperative VAS and postoperative opioid consumption were recorded. RESULTS: Duration of block was significantly longer in QLB-3 group when compared to QLB-2 group (20.1 + 6.2 h versus 12.0 + 4.8 respectively) with P value of < 0.001. A statistically significant lower VAS score was recorded in QLB-3 group immediately and 12 h postoperative. QLB-3 group showed a statistically significant delayed time of first analgesic request and less postoperative morphine consumption with P value of < 0.001 and 0.001 respectively. CONCLUSIONS: Ultrasound guided postsurgical transmuscular approach of QLB (QLB-3) using 20 ml 0.25% bupivacaine produces more postoperative analgesic effect and less postoperative opioid consumption when compared to posterior QLB approach (QLB-2) in patients underwent unilateral inguinal hernia repair under general anesthesia. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03526731 - on 16 May 2018.

Heat Shock Proteins in the "Hot" Mitochondrion: Identity and Putative Roles.

The mitochondrion is known as the "powerhouse" of eukaryotic cells since it is the main site of adenosine 5'-triphosphate (ATP) production. Using a temperature-sensitive fluorescent probe, it has recently been suggested that the stray free energy, not captured into ATP, is potentially sufficient to sustain mitochondrial temperatures higher than the cellular environment, possibly reaching up to 50 °C. By 50 °C, some DNA and mitochondrial proteins may reach their melting temperatures; how then do these biomolecules maintain their structure and function? Further, the production of reactive oxygen species (ROS) accelerates with temperature, implying higher oxidative stresses in the mitochondrion than generally appreciated. Herein, it is proposed that mitochondrial heat shock proteins (particularly Hsp70), in addition to their roles in protein transport and folding, protect mitochondrial proteins and DNA from thermal and ROS damage. Other thermoprotectant mechanisms are also discussed.

Warfarin-induced spontaneous retroperitoneal hemorrhage from the renal vein: A rare case with an uncommon etiology.

The overall rate of major bleeding in patients with atrial fibrillation receiving warfarin therapy is approximately 4%. Among these 4% patients, spontaneous retroperitoneal hemorrhage (SRH) is a rare but potentially lethal complication with a nonspecific presentation that can lead to missed or delayed diagnosis. The current literature provides little direction for diagnosis and management of such cases. Anticoagulation-related SRH is associated with a high mortality rate (approximately 20%). Despite the vague presentation, prompt diagnosis is crucial to reverse the anticoagulation and prevent further bleeding. Contrast-enhanced computed tomography (CT) of the abdomen is the imaging modality of choice in suspected cases. Patients with SRH require aggressive treatment with blood transfusions, interventional radiological procedures, percutaneous drainage or surgical evacuation of the hematoma. We report a case of warfarin-induced SRH from the renal vein in a patient who presented to our emergency department with acute, nonspecific abdominal pain and shock. We diagnosed the patient with warfarin-induced SRH on the basis of clinical suspicion and characteristic CT findings. We initially treated the patient conservatively, followed by embolization of the right renal artery during the late course of hospital stay, and he was discharged with good recovery. SRH should be considered in the differential diagnosis of abdominal pain, hypotension, and/or decreased hemoglobin levels in patients receiving anticoagulation therapy, especially in those with preexisting end-stage renal disease.

A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host.

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.

Telomerase reverse transcriptase coordinates with the epithelial-to-mesenchymal transition through a feedback loop to define properties of breast cancer stem cells.

Telomerase and its core component, telomerase reverse transcriptase (hTERT), are critical for stem cell compartment integrity. Normal adult stem cells have the longest telomeres in a given tissue, a property mediated by high hTERT expression and high telomerase enzymatic activity. In contrast, cancer stem cells (CSCs) have short telomeres despite high expression of hTERT, indicating that the role of hTERT in CSCs is not limited to telomere elongation and/or maintenance. The function of hTERT in CSCs remains poorly understood. Here, we knocked down hTERT expression in CSCs and observed a morphological shift to a more epithelial phenotype, suggesting a role for hTERT in the epithelial-to-mesenchymal transition (EMT) of CSCs. Therefore, in this study, we systematically explored the relationship between hTERT and EMT and identified a reciprocal, bi-directional feedback loop between hTERT and EMT in CSCs. We found that hTERT expression is mutually exclusive to the mesenchymal phenotype and that, reciprocally, loss of the mesenchymal phenotype represses hTERT expression. We also showed that hTERT plays a critical role in the expression of key CSC markers and nuclear ß-catenin localization, increases the percentage of cells with side-population properties, and upregulates the CD133 expression. hTERT also promotes chemoresistance properties, tumorsphere formation and other important functional CSC properties. Subsequently, hTERT knockdown leads to the loss of the above advantages, indicating a loss of CSC properties. Our findings suggest that targeting hTERT might improve CSCs elimination by transitioning them from the aggressive mesenchymal state to a more steady epithelial state, thereby preventing cancer progression.

Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins.

Multiple species within the Acinetobacter genus are nosocomial opportunistic pathogens of increasing relevance worldwide. Among the virulence factors utilized by these bacteria are the type IV pili and a protein O-glycosylation system. Glycosylation is mediated by O-oligosaccharyltransferases (O-OTases), enzymes that transfer the glycan from a lipid carrier to target proteins. O-oligosaccharyltransferases are difficult to identify due to similarities with the WaaL ligases that catalyze the last step in lipopolysaccharide synthesis. A bioinformatics analysis revealed the presence of two genes encoding putative O-OTases or WaaL ligases in most of the strains within the genus Acinetobacter. Employing A. nosocomialis M2 and A. baylyi ADP1 as model systems, we show that these genes encode two O-OTases, one devoted uniquely to type IV pilin, and the other one responsible for glycosylation of multiple proteins. With the exception of ADP1, the pilin-specific OTases in Acinetobacter resemble the TfpO/PilO O-OTase from Pseudomonas aeruginosa. In ADP1 instead, the two O-OTases are closely related to PglL, the general O-OTase first discovered in Neisseria. However, one of them is exclusively dedicated to the glycosylation of the pilin-like protein ComP. Our data reveal an intricate and remarkable evolutionary pathway for bacterial O-OTases and provide novel tools for glycoengineering.

GPx-1 modulates Akt and P70S6K phosphorylation and Gadd45 levels in MCF-7 cells.

Selenium has been shown to prevent cancer in animal models, and recent data indicate it is likely to be effective in humans as well. One selenium-containing protein, the cytoplasmic form of glutathione peroxidase (GPx-1), has been implicated in cancer risk and development by genetic studies identifying at-risk alleles and loss of heterozygosity in tumors. In order to evaluate the biological consequences of GPx-1 overexpression, human MCF-7 cells were stably transfected with a GPx-1 expression construct and the effects of GPx-1 on protein kinases associated with stress responses were determined. GPx-1 overexpression affected phosphorylation of p70S6K, whereas Erk1/2 and p38 MAPK were not affected. Site-specific phosphorylation of Akt declined and the levels of Gadd45, a DNA damage response protein, increased significantly as a consequence of elevated GPx-1 expression. Effects on p70S6K and Gadd45 after selenium supplementation have been reported, and given previous data demonstrating a role for GPx-1 in cancer etiology, these results support the concept that the chemopreventive properties of selenium may be due, at least in part, to its role in regulating GPx-1.