Inhibition of NRF2 signaling overcomes acquired resistance to arsenic trioxide in FLT3-mutated Acute Myeloid Leukemia.

De novo acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) have worse treatment outcomes. Arsenic trioxide (ATO) used in the treatment of acute promyelocytic leukemia (APL) has been reported to be effective in degrading the FLT3 protein in AML cell lines and sensitizing non-APL AML patient samples in-vitro. We have previously reported that primary cells from FLT3-ITD mutated AML patients were sensitive to ATO in-vitro compared to other non-M3 AML and molecular/pharmacological inhibition of NF-E2 related factor 2 (NRF2), a master regulator of antioxidant response improved the chemosensitivity to ATO and daunorubicin even in non FLT3-ITD mutated cell lines and primary samples. We examined the effects of molecular/pharmacological suppression of NRF2 on acquired ATO resistance in the FLT3-ITD mutant AML cell line (MV4-11-ATO-R). ATO-R cells showed increased NRF2 expression, nuclear localization, and upregulation of bonafide NRF2 targets. Molecular inhibition of NRF2 in this resistant cell line improved ATO sensitivity in vitro. Digoxin treatment lowered p-AKT expression, abrogating nuclear NRF2 localization and sensitizing cells to ATO. However, digoxin and ATO did not sensitize non-ITD AML cell line THP1 with high NRF2 expression. Digoxin decreased leukemic burden and prolonged survival in MV4-11 ATO-R xenograft mice. We establish that altering NRF2 expression may reverse acquired ATO resistance in FLT3-ITD AML.

A comprehensive SARS-CoV-2 and COVID-19 review, Part 2: host extracellular to systemic effects of SARS-CoV-2 infection.

COVID-19, the disease caused by SARS-CoV-2, has caused significant morbidity and mortality worldwide. The betacoronavirus continues to evolve with global health implications as we race to learn more to curb its transmission, evolution, and sequelae. The focus of this review, the second of a three-part series, is on the biological effects of the SARS-CoV-2 virus on post-acute disease in the context of tissue and organ adaptations and damage. We highlight the current knowledge and describe how virological, animal, and clinical studies have shed light on the mechanisms driving the varied clinical diagnoses and observations of COVID-19 patients. Moreover, we describe how investigations into SARS-CoV-2 effects have informed the understanding of viral pathogenesis and provide innovative pathways for future research on the mechanisms of viral diseases.

A Comparative Study of Intrathecal Fentanyl and Nalbuphine as an Adjuvant to Hyperbaric Bupivacaine for Spinal Anesthesia in Lower Limb Orthopedic Surgeries: A Prospective, Double-Blind, Randomized Controlled Study.

BACKGROUND: Spinal anaesthesia is the most commonly used technique for lower limb orthopaedic surgeries as it is economical and easy to administer. Opioids as adjuvants to local anaesthetics during spinal anaesthesia have played a vital role in reducing post-operative pain qualitatively and effectively. METHODS: This prospective randomised study was conducted on 100 patients divided into two groups scheduled for lower limb orthopaedic surgeries. Group bupivacaine fentanyl (BF) received 25 mcg of fentanyl with 15 mg of bupivacaine and Group bupivacaine nalbuphine (BN) received 1 mg of nalbuphine and 15 mg of 0.5% bupivacaine. The aim of the study was to compare the analgesic efficacy of intrathecal fentanyl and nalbuphine as an adjuvant to hyperbaric bupivacaine for spinal anaesthesia. Duration of effective analgesia, haemodynamic parameters, onset and duration of sensory and motor block, adverse effects, and visual analogue scale (VAS) score were assessed. RESULTS: Duration of effective analgesia was 388±24.88 minutes in the BN group and was higher (p-value <0.001) in comparison to the BF group, which was 304.70±15.76 minutes. CONCLUSION: Nalbuphine was more effective than fentanyl in providing post-operative analgesia when used as an adjuvant to hyperbaric bupivacaine.

Robust Response of the Clinical Laboratory to the COVID-19 Pandemic despite Significant Challenges.

BACKGROUND: Clinical laboratories immediately provided rapid, reliable, and high-throughout diagnostic testing for COVID-19, which was an essential component in combating the pandemic. As the pandemic evolved, the clinical laboratory was faced with additional challenges. However, there are limited studies on the impact of the pandemic on the clinical laboratory over the past 3 years. METHODS: The American Association for Clinical Chemistry (AACC) sent 8 surveys over a 32-month time period to international clinical laboratory leadership asking questions about COVID-19 testing, supplies, staffing, and lessons learned. RESULTS: There were a total of 191 unique respondents: 133 laboratories in the US and 58 laboratories from 37 other countries participated. By May 2020, more than 70% of laboratories offered COVID-19 diagnostic testing with average turnaround times ranging from 1 to 24 h. Daily COVID-19 testing volumes peaked in January of 2022 at a median of 775 tests per day. Throughout the pandemic, supplies and staffing concerns increased. In most of the 8 surveys, 55% to 65% of laboratories reported they were unable to obtain supplies. Obtaining reagents and test kits was the most problematic. Staffing challenges continue to be a significant concern and most laboratories have struggled hiring testing personnel. CONCLUSIONS: Survey results were utilized to demonstrate the impact of the pandemic on the clinical laboratory community, and importantly, findings were presented to the White House Coronavirus Taskforce. Overall, the clinical laboratories had a robust response to the COVID-19 pandemic, and despite ongoing and evolving challenges, continue to provide rapid diagnostic testing.

Comparative evaluation of bupivacaine with magnesium sulphate and dexamethasone as adjuvants in ultrasound-guided transversus abdominis plane block for open unilateral inguinal hernia surgeries: A randomised controlled trial.

Background and Aims: Inguinal hernia repair is associated with moderate to severe pain that is most extreme in the first 24 hours. The aim of this study was to compare the efficacy of dexamethasone versus magnesium sulphate (MgSO4) with bupivacaine in ultrasound-guided transversus abdominis plane (TAP) block for patients undergoing unilateral inguinal hernioplasty. Methods: Eighty patients were randomly allocated to two groups to receive ultrasound-guided TAP block postoperatively with either 20 ml of 0.25% bupivacaine with 8 mg of dexamethasone (Group BD) or 20 ml of 0.25% bupivacaine with 250 mg of MgSO4 (Group BM). Patients were assessed for the first 24 hours after surgery for pain at rest and movement using a numerical rating scale (NRS). Two mg/kg of tramadol was administered as rescue analgesia. The time to first demand tramadol, total consumption of tramadol, patient satisfaction score and side effects were evaluated. Results: The time to the first dose of rescue analgesia was significantly longer in BD group (596.13 ± 57.93 min) than in the BM group (422.50 ± 51.95 min). The NRS scores in the BD group were significantly lower compared to the BM group both at rest and on movement. The total requirement of tramadol was significantly less in the BD group (154.55 ± 59.11 mg) compared to the BM group (270.25 ± 105.72 mg). The incidence of side effects was lower and patient satisfaction was higher in BD group compared to BM group. Conclusion: Bupivacaine with dexamethasone in TAP block after unilateral open inguinal hernioplasty provides increased duration of analgesia and decreased requirement for rescue analgesics compared to magnesium sulphate, with lesser side effects and better patient satisfaction.

Modulating retinoid-X-receptor alpha (RXRA) expression sensitizes chronic myeloid leukemia cells to imatinib in vitro and reduces disease burden in vivo.

Introduction: The ligand-activated transcription factors, nuclear hormone receptors (NHRs), remain unexplored in hematological malignancies except for retinoic acid receptor alpha (RARA). Methods: Here we profiled the expression of various NHRs and their coregulators in Chronic myeloid leukemia (CML) cell lines and identified a significant differential expression pattern between inherently imatinib mesylate (IM)-sensitive and resistant cell lines. Results: Retinoid-X-receptor alpha (RXRA) was downregulated in CML cell lines inherently resistant to IM and in primary CML CD34+ cells. Pre-treatment with clinically relevant RXRA ligands improved sensitivity to IM in-vitro in both CML cell lines and primary CML cells. This combination effectively reduced the viability and colony-forming capacity of CML CD34+ cells in-vitro. In-vivo, this combination reduced leukemic burden and prolonged survival. Overexpression (OE) of RXRA inhibited proliferation and improved sensitivity to IM in-vitro. In-vivo, RXRA OE cells showed reduced engraftment of cells in the bone marrow, improved sensitivity to IM, and prolonged survival. Both RXRA OE and ligand treatment markedly reduced BCR::ABL1 downstream kinase activation, activating apoptotic cascades and improving sensitivity to IM. Importantly, RXRA OE also led to the disruption of the oxidative capacity of these cells. Conclusion: Combining IM with clinically available RXRA ligands could form an alternative treatment strategy in CML patients with suboptimal response to IM.

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Peripheral Nerve Stimulator Versus Ultrasound-Guided Femoral Nerve Block for Knee Arthroscopy Procedures: A Randomized Controlled Trial.

Background Femoral nerve block (FNB) provides effective analgesia and is a widely used technique for postoperative pain relief for orthopedic procedures on lower limbs. This study aims to compare the efficacy of ultrasonography (USG) versus peripheral nerve stimulator (PNS)-guided FNB in knee arthroscopic procedures. Methodology This randomized comparative study included two study groups with 30 participants in each group who were given FNB with either PNS or USG for knee arthroscopic procedures following spinal anesthesia. The study evaluated the number of needle repositioning, the time taken for performing the block, the efficacy of postoperative analgesia based on the duration of the block, and patient satisfaction. Results The number of needle repositioning and time taken to finish the procedure using USG was lower compared to the group using PNS (p < 0.001). The duration of the block was comparable in both groups (p = 0.584). Patients were satisfied with both techniques and responded as either very good or outstanding and chose neither as inferior (p = 0.310). Conclusions Both techniques have equal efficacy concerning the duration of the effect of the block and patient satisfaction. However, the procedural time and number of needle repositioning were significantly less in the group where USG was used for the block.

Challenges and considerations for single-cell and spatially resolved transcriptomics sample collection during spaceflight.

Single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (SRT) have experienced rapid development in recent years. The findings of spaceflight-based scRNA-seq and SRT investigations are likely to improve our understanding of life in space and our comprehension of gene expression in various cell systems and tissue dynamics. However, compared to their Earth-based counterparts, gene expression experiments conducted in spaceflight have not experienced the same pace of development. Out of the hundreds of spaceflight gene expression datasets available, only a few used scRNA-seq and SRT. In this perspective piece, we explore the growing importance of scRNA-seq and SRT in space biology and discuss the challenges and considerations relevant to robust experimental design to enable growth of these methods in the field.

Perfusion Index and Its Correlation With Intraoperative Hypotension in Lower-Segment Cesarean Section Under Spinal Anesthesia: A Prospective Observational Study in a Tertiary Care Hospital in Eastern India.

Background Hypotension is commonly encountered in patients undergoing lower-segment cesarean section (LSCS) under the subarachnoid block (SAB) owing to decreased vascular resistance caused by the sympathetic blockade and decreased cardiac output because of blood pooling in blocked areas of the body. Perfusion index (PI) is a good indicator of systemic vascular resistance and can foretell hypotension. This study aimed to associate baseline PI with intraoperative hypotension after SAB in LSCS. Methodology This was a prospective observational study with a sample size of 50. The baseline PI was recorded every 10 seconds for one minute in a supine position on the right index finger at room temperature of 26°C to 28°C. The blood pressure (BP) and heart rate (HR) were recorded at an interval of one minute for three minutes. The mean of PI, BP, and HR were taken as the preoperative value. Spinal anesthesia was administered as per institutional protocol. Hypotension, defined as mean arterial pressure (MAP) <20% of baseline or MAP <60 mmHg was treated with vasopressors. Regression analysis with the Spearman correlation coefficient was done to correlate PI and hypotension. Results The incidence of hypotension in parturients with PI <2.85 was 28.6% (5/20) and in parturients with PI >2.85 was 82.8% (p < 0.001). The requirement of sympathomimetics was higher in parturients with PI >2.85.The area under the receiver operating characteristic curve was 0.8883. A cut-off PI value of 2.85 can identify parturients at risk for central neuraxial block-induced hypotension with a sensitivity of 80% and a specificity of 75% (p < 0.001). Conclusions The PI is a useful tool for predicting hypotension in healthy parturients undergoing elective cesarean section under SAB.

AACC Guidance Document on the Use of Point-of-Care Testing in Fertility and Reproduction.

BACKGROUND: The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. METHODS: A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. RESULTS: Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. CONCLUSIONS: This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype.

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

Extraterrestrial Gynecology: Could Spaceflight Increase the Risk of Developing Cancer in Female Astronauts? An Updated Review.

Outer space is an extremely hostile environment for human life, with ionizing radiation from galactic cosmic rays and microgravity posing the most significant hazards to the health of astronauts. Spaceflight has also been shown to have an impact on established cancer hallmarks, possibly increasing carcinogenic risk. Terrestrially, women have a higher incidence of radiation-induced cancers, largely driven by lung, thyroid, breast, and ovarian cancers, and therefore, historically, they have been permitted to spend significantly less time in space than men. In the present review, we focus on the effects of microgravity and radiation on the female reproductive system, particularly gynecological cancer. The aim is to provide a summary of the research that has been carried out related to the risk of gynecological cancer, highlighting what further studies are needed to pave the way for safer exploration class missions, as well as postflight screening and management of women astronauts following long-duration spaceflight.

The interplay between lncRNAs, RNA-binding proteins and viral genome during SARS-CoV-2 infection reveals strong connections with regulatory events involved in RNA metabolism and immune response.

Rationale: Viral infections are complex processes based on an intricate network of molecular interactions. The infectious agent hijacks components of the cellular machinery for its profit, circumventing the natural defense mechanisms triggered by the infected cell. The successful completion of the replicative viral cycle within a cell depends on the function of viral components versus the cellular defenses. Non-coding RNAs (ncRNAs) are important cellular modulators, either promoting or preventing the progression of viral infections. Among these ncRNAs, the long non-coding RNA (lncRNA) family is especially relevant due to their intrinsic functional properties and ubiquitous biological roles. Specific lncRNAs have been recently characterized as modulators of the cellular response during infection of human host cells by single stranded RNA viruses. However, the role of host lncRNAs in the infection by human RNA coronaviruses such as SARS-CoV-2 remains uncharacterized. Methods: In the present work, we have performed a transcriptomic study of a cohort of patients with different SARS-CoV-2 viral load and analyzed the involvement of lncRNAs in supporting regulatory networks based on their interaction with RNA-binding proteins (RBPs). Results: Our results revealed the existence of a SARS-CoV-2 infection-dependent pattern of transcriptional up-regulation in which specific lncRNAs are an integral component. To determine the role of these lncRNAs, we performed a functional correlation analysis complemented with the study of the validated interactions between lncRNAs and RBPs. This combination of in silico functional association studies and experimental evidence allowed us to identify a lncRNA signature composed of six elements - NRIR, BISPR, MIR155HG, FMR1-IT1, USP30-AS1, and U62317.2 - associated with the regulation of SARS-CoV-2 infection. Conclusions: We propose a competition mechanism between the viral RNA genome and the regulatory lncRNAs in the sequestering of specific RBPs that modulates the interferon response and the regulation of RNA surveillance by nonsense-mediated decay (NMD).

An evaluation of liver function tests in severe acute respiratory syndrome - Corona virus 2 (SARS-CoV-2) infection in the backdrop of chronic kidney disease.

Background: The SARS-CoV-2 pandemic has emerged as the most challenging global health problem of this century. The concomitant presence of co-morbidities like chronic kidney disease (CKD), diabetes, CHD, further complicates the problem. Aim: To assess the patterns of LFT abnormalities in patients of SARS-CoV-2 infection with and without CKD and evaluate the probable outcomes. Materials and Methods: A cross-sectional retrospective observational study done on 600 patient samples (Group 1: SARS-CoV-2 without CKD, Group 2: SARS-CoV-2 with CKD and Group 3: CKD uninfected with SARS-CoV-2) which were processed for LFT and KFT. Results: AST and ALT were significantly higher in all SARS-CoV-2 infected; Group 1 mean ± 2SD, (63.63 ± 42.89U/L & 50.25 ± 46.53U/L), group 2 (90.59 ± 62.51U/L & 72.09 ± 67.24 U/L) as compared to Group 3 (25.24 ± 7.47U/L & 24.93 ± 11.44U/L). A statistically significant elevation is seen in these two parameters in Group 2 as compared to Group 1. There was a negative significant correlation between eGFR and AST/ALT levels in Group 1 (p < 0.05). In Group 2, a weak positive correlation was seen with ALT. Group 3, eGFR's showed strong correlations with AST and ALT levels; reduction in kidney function correlated well with increase in serum ALP levels. Conclusions: This study establishes that SARS-CoV-2 infected, with CKD, show higher elevations in serum aminotransferase levels in comparison to those without CKD. In contrast, the CKD group not infected, shows a decline in serum aminotransferase levels. Serum ALT values in SARS-CoV-2 show significant correlation with eGFR. Also, elevated ALP values in CKD patients may be used as an indicator of declining kidney function.

Procedural Sedation Using Two Different Proportions of Ketamine-Propofol Combination in Short Gynecological Procedures: A Randomized Controlled Trial.

BACKGROUND: Procedural sedation with a combination of propofol and ketamine for short-duration surgeries is a convenient technique of anesthesia as it has a faster recovery avoiding the side effects of general anesthesia. The aim of this study was to compare the sedative and analgesic effects of two different proportions of ketamine and propofol combination in patients undergoing short gynecological procedures. METHODS: A randomized double-blind study was conducted in 140 patients posted for elective gynecological procedures with a duration equal to or less than 30 minutes. After premedication of all participants, sedation was induced with bolus administration (0.1 mL/kg) of the study drugs to achieve desired Ramsay sedation score (RSS) of 6, followed by infusion at 0.3 mL/kg/h (Group A, ketamine:propofol in the ratio of 1:4 and Group B, ketamine:propofol in the ratio of 1:2). The adequacy of sedation, volume of drug to induce the patient, time to achieve desired RSS, time for first bolus dose, the total volume of the drugs, hemodynamic variables, awakening time, and side effects were observed. RESULTS: The incidence of movement of lower extremities was found to be significantly lower in the higher concentration ketamine group (Group B, P - 0.028). The volume of a drug for induction and the duration to reach RSS of 6 were significantly lower in Group B with P-values of 0.002 and <0.001, respectively. Hemodynamic variables, awakening time, and side effects were not statistically significant between the two groups. CONCLUSION: Ketamine-propofol combination in the ratio 1:2 provides better sedation and analgesia with no increased side-effects compared to ketamine-propofol in the ratio 1:4 for short outpatient gynecological procedures.

Clinicians' Probability Calculator to Convert Pre-Test to Post-Test Probability of SARS-CoV-2 Infection Based on Method Validation from Each Laboratory.

BACKGROUND: Despite best efforts, false positive and false negative test results for SARS-CoV-2 are unavoidable. Likelihood ratios convert a clinical opinion of pre-test probability to post-test probability, independently of prevalence of disease in the test population. METHODS: The authors examined results of PPA (Positive Percent Agreement, sensitivity) and NPA (Negative Percent Agreement, specificity) from 73 laboratory experiments for molecular tests for SARS-CoV-2 as reported to the FIND database, and for two manufacturers' claims in FDA EUA submissions.PPA and NPA were converted to likelihood ratios to calculate post-test probability of disease based on clinical opinion of pre-test probability. Confidence intervals were based on the number of samples tested. An online calculator was created to help clinicians identify false-positive, or false-negative SARS-CoV-2 test results for COVID-19 disease. RESULTS: Laboratory results from the same test methods did not mirror each other or the manufacturer. Laboratory studies showed PPA from 17% to 100% and NPA from 70.4% to 100%. The number of known samples varied 8 to 675 known patient samples, which greatly impacted confidence intervals. CONCLUSION: Post-test probability of the presence of disease (true-positive or false-negative tests) varies with clinical pre-test probability, likelihood ratios and confidence intervals.The Clinician's Probability Calculator creates reports to help clinicians estimate post-test probability of COVID-19 based on the testing laboratory's verified PPA and NPA.