Evidence for the evolving role of neoadjuvant and perioperative immunotherapy in resectable non-small cell lung cancer.

The treatment of early-stage non-small cell lung cancer (NSCLC) is becoming increasingly complex. Standard of care management for the past decade has been adjuvant chemotherapy following curative intent resection regardless of nodal status or tumour profile. With the increased incorporation of immunotherapy in NSCLC, especially in the locally advanced, unresectable, or metastatic settings, multiple studies have sought to assess its utility in early-stage disease. While there are suboptimal responses to neoadjuvant chemotherapy alone, there is a strong rationale for the use of neoadjuvant immunotherapy in tumour downstaging, based upon the concept of enhanced T cell priming at the time of a high tumour antigen burden, and demonstrated clinically in other solid tumours, such as melanoma. In the NSCLC cancer setting, currently over 20 combinations of chemoimmunotherapy in the neoadjuvant and perioperative setting have been studied with results variable. Multiple large phase III studies have demonstrated that neoadjuvant chemoimmunotherapy combinations result in significant advances in pathological response, disease free and overall survival which has led to practice change across the world. Currently, combination immunotherapy regimens with novel agents targeting alternate immunomodulatory pathways are now being investigated. Given this, the landscape of treatment in resectable early-stage NSCLC has become increasingly complex. This review outlines the literature of neoadjuvant and perioperative immunotherapy and discusses its potential benefits and complexities and ongoing considerations into future research.

Loss of glucose-stimulated ß-cell Nr4a1 expression impairs insulin secretion and glucose homeostasis.

A central aspect of type 2 diabetes is decreased functional ß-cell mass. The orphan nuclear receptor Nr4a1 is critical for fuel utilization, but little is known regarding its regulation and function in the ß-cell. Nr4a1 expression is decreased in type 2 diabetes rodent ß-cells and type 2 diabetes patient islets. We have shown that Nr4a1-deficient mice have reduced ß-cell mass and that Nr4a1 knockdown impairs glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 ß-cells. Here, we demonstrate that glucose concentration directly regulates ß-cell Nr4a1 expression. We show that 11 mM glucose increases Nr4a1 expression in INS-1 832/13 ß-cells and primary mouse islets. We show that glucose functions through the cAMP/PKA/CREB pathway to regulate Nr4a1 mRNA and protein expression. Using Nr4a1-/- animals, we show that Nr4a1 is necessary for GSIS and systemic glucose handling. Using RNA-seq, we define Nr4a1-regulated pathways in response to glucose in the mouse islet, including Glut2 expression. Our data suggest that Nr4a1 plays a critical role in the ß-cells response to the fed state.NEW & NOTEWORTHY Nr4a1 has a key role in fuel metabolism and ß-cell function, but its exact role is unclear. Nr4a1 expression is regulated by glucose concentration using cAMP/PKA/CREB pathway. Nr4a1 regulates Glut2, Ndufa4, Ins1, In2, Sdhb, and Idh3g expression in response to glucose treatment. These results suggest that Nr4a1 is necessary for proper insulin secretion both through glucose uptake and metabolism machinery.

Zebrafish: unraveling genetic complexity through duplicated genes.

The zebrafish is an invaluable model organism for genetic, developmental, and disease research. Although its high conservation with humans is often cited as justification for its use, the zebrafish harbors oft-ignored genetic characteristics that may provide unique insights into gene structure and function. Zebrafish, along with other teleost fish, underwent an additional round of whole genome duplication after their split from tetrapods-resulting in an abundance of duplicated genes when compared to other vertebrates. These duplicated genes have evolved in distinct ways over the ensuing 350 million years. Thus, each gene within a duplicated gene pair has nuanced differences that create a unique identity. By investigating both members of the gene pair together, we can elucidate the mechanisms that underly protein structure and function and drive the complex interplay within biological systems, such as signal transduction cascades, genetic regulatory networks, and evolution of tissue and organ function. It is crucial to leverage such studies to explore these molecular dynamics, which could have far-reaching implications for both basic science and therapeutic development. Here, we will review the role of gene duplications and the existing models for gene divergence and retention following these events. We will also highlight examples within each of these models where studies comparing duplicated genes in the zebrafish have yielded key insights into protein structure, function, and regulation.

Valproic Acid Causes Redox-Regulated Post-Translational Protein Modifications That Are Dependent upon P19 Cellular Differentiation States.

Valproic acid (VPA) is a common anti-epileptic drug and known neurodevelopmental toxicant. Although the exact mechanism of VPA toxicity remains unknown, recent findings show that VPA disrupts redox signaling in undifferentiated cells but has little effect on fully differentiated neurons. Redox imbalances often alter oxidative post-translational protein modifications and could affect embryogenesis if developmentally critical proteins are targeted. We hypothesize that VPA causes redox-sensitive post-translational protein modifications that are dependent upon cellular differentiation states. Undifferentiated P19 cells and P19-derived neurons were treated with VPA alone or pretreated with D3T, an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant pathway, prior to VPA exposure. Undifferentiated cells treated with VPA alone exhibited an oxidized glutathione redox couple and increased overall protein oxidation, whereas differentiated neurons were protected from protein oxidation via increased S-glutathionylation. Pretreatment with D3T prevented the effects of VPA exposure in undifferentiated cells. Taken together, our findings support redox-sensitive post-translational protein alterations in undifferentiated cells as a mechanism of VPA-induced developmental toxicity and propose NRF2 activation as a means to preserve proper neurogenesis.

RNA Sequencing Demonstrates Ex Vivo Neocortical Transcriptomic Changes Induced by Epileptiform Activity in Male and Female Mice.

Seizures are generally associated with epilepsy but may also be a symptom of many other neurological conditions. A hallmark of a seizure is the intensity of the local neuronal activation, which can drive large-scale gene transcription changes. Such changes in the transcriptional profile likely alter neuronal function, thereby contributing to the pathological process. Therefore, there is a strong clinical imperative to characterize how gene expression is changed by seizure activity. To this end, we developed a simplified ex vivo technique for studying seizure-induced transcriptional changes. We compared the RNA sequencing profile in mouse neocortical tissue with up to 3 h of epileptiform activity induced by 4-aminopyridine (4AP) relative to control brain slices not exposed to the drug. We identified over 100 genes with significantly altered expression after 4AP treatment, including multiple genes involved in MAPK, TNF, and neuroinflammatory signaling pathways, all of which have been linked to epilepsy previously. Notably, the patterns in male and female brain slices were almost identical. Various immediate early genes were among those showing the largest upregulation. The set of down-regulated genes included ones that might be expected either to increase or to decrease neuronal excitability. In summary, we found the seizure-induced transcriptional profile complex, but the changes aligned well with an analysis of published epilepsy-associated genes. We discuss how simple models may provide new angles for investigating seizure-induced transcriptional changes.

Heterochronic shift in gene expression leads to ontogenetic morphological divergence between two closely related polyploid species.

Heterochrony-alteration to the rate or timing of development-is an important mechanism of trait differentiation associated with speciation. Heterochrony may explain the morphological divergence between two polyploid species, June sucker (Chasmistes liorus) and Utah sucker (Catostomus ardens). The larvae of both species have terminal mouths; however, as adults, June sucker and Utah sucker develop subterminal and ventral mouths, respectively. We document a difference in the timing of shape development and a corresponding change in the timing of gene expression, suggesting the distinctive mouth morphology in June suckers may result from paedomorphosis. Specifically, adult June suckers exhibit an intermediate mouth morphology between the larval (terminal) and ancestral (ventral) states. Endemic and sympatric Chasmistes/Catostomus pairs in two other lakes also are morphologically divergent, but genetically similar. These species pairs could have resulted from the differential expression of genes and corresponding divergence in trait development. Paedomorphosis may lead to adaptive diversification in Catostomids.

Detection of neuron-derived cfDNA in blood plasma: a new diagnostic approach for neurodegenerative conditions.

Neurodegenerative diseases, such as Alzheimer's disease (AD), pose significant challenges in early diagnosis, leading to irreversible brain damage and cognitive decline. In this study, we present a novel diagnostic approach that utilizes whole molecule analysis of neuron-derived cell-free DNA (cfDNA) as a biomarker for early detection of neurodegenerative diseases. By analyzing Differential Methylation Regions (DMRs) between purified cortical neurons and blood plasma samples, we identified robust biomarkers that accurately distinguish between neuronal and non-neuronal cfDNA. The use of cfDNA offers the advantage of convenient and minimally invasive sample collection compared to traditional cerebrospinal fluid or tissue biopsies, making this approach more accessible and patient friendly. Targeted sequencing at the identified DMR locus demonstrated that a conservative cutoff of 5% of neuron-derived cfDNA in blood plasma accurately identifies 100% of patients diagnosed with AD, showing promising potential for early disease detection. Additionally, this method effectively differentiated between patients with mild cognitive impairment (MCI) who later progressed to AD and those who did not, highlighting its prognostic capabilities. Importantly, the differentiation between patients with neurodegenerative diseases and healthy controls demonstrated the specificity of our approach. Furthermore, this cfDNA-based diagnostic strategy outperforms recently developed protein-based assays, which often lack accuracy and convenience. While our current approach focused on a limited set of loci, future research should explore the development of a more comprehensive model incorporating multiple loci to increase diagnostic accuracy further. Although certain limitations, such as technical variance associated with PCR amplification and bisulfite conversion, need to be addressed, this study emphasizes the potential of cfDNA analysis as a valuable tool for pre-symptomatic detection and monitoring of neurodegenerative diseases. With further development and validation, this innovative diagnostic strategy has the potential to significantly impact the field of neurodegenerative disease research and patient care, offering a promising avenue for early intervention and personalized therapeutic approaches.

The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Effect of chemotherapy on the uterus of young adult cancer survivors.

Objective: To investigate the impact of chemotherapy on the uterus. Design: Cross-sectional pilot study. Setting: Single university fertility clinic. Patients: Twelve patients with a history of alkylating agent chemotherapy exposure after Hodgkin lymphoma (cancer) vs. 12 normally menstruating women (controls). Interventions: The inclusion criteria were age of 18-45 years and consent for endometrial biopsy. The exclusion criteria were the absence of the uterus, completed pelvic radiation, uterine or cervical cancer, and metastatic cancer. Each participant underwent endometrial biopsy and pelvic ultrasound. All study visits were conducted in the late proliferative phase of the menstrual cycle. Main Outcome Measures: Uterine volume, blood flow, endometrial thickness, histology, deoxyribonucleic acid methylation pattern, and relative ribonucleic acid (RNA) expression level during the same phase of the menstrual cycle. Results: In the study group, visits were conducted at a median of 31.5 (13.5-42.5) months after chemotherapy. The median uterine volume among cancer survivors was 36 (11.3-67) cm3, and that of the general population controls was 39 (13-54) cm3. On histologic examination, there were no cytologic or architectural atypia. The RNA-sequencing analysis revealed poor clustering of both control and treatment samples. However, we identified 3 differentially expressed genes on RNA-sequencing, but there was no concordance found among the differentially expressed genes and deoxyribonucleic acid methylation changes suggesting most likely false-positive results. Conclusions: Approximately 2.5 years after chemotherapy, a time at which several survivors of Hodgkin lymphoma may resume family-building, endometrial thickness and endometrial histology were not significantly affected by a history of alkylating agent chemotherapy exposure.

Single-Cell RNA Sequencing Characterizes the Molecular Heterogeneity of the Larval Zebrafish Optic Tectum.

The optic tectum (OT) is a multilaminated midbrain structure that acts as the primary retinorecipient in the zebrafish brain. Homologous to the mammalian superior colliculus, the OT is responsible for the reception and integration of stimuli, followed by elicitation of salient behavioral responses. While the OT has been the focus of functional experiments for decades, less is known concerning specific cell types, microcircuitry, and their individual functions within the OT. Recent efforts have contributed substantially to the knowledge of tectal cell types; however, a comprehensive cell catalog is incomplete. Here we contribute to this growing effort by applying single-cell RNA Sequencing (scRNA-seq) to characterize the transcriptomic profiles of tectal cells labeled by the transgenic enhancer trap line y304Et(cfos:Gal4;UAS:Kaede). We sequenced 13,320 cells, a 4X cellular coverage, and identified 25 putative OT cell populations. Within those cells, we identified several mature and developing neuronal populations, as well as non-neuronal cell types including oligodendrocytes and microglia. Although most mature neurons demonstrate GABAergic activity, several glutamatergic populations are present, as well as one glycinergic population. We also conducted Gene Ontology analysis to identify enriched biological processes, and computed RNA velocity to infer current and future transcriptional cell states. Finally, we conducted in situ hybridization to validate our bioinformatic analyses and spatially map select clusters. In conclusion, the larval zebrafish OT is a complex structure containing at least 25 transcriptionally distinct cell populations. To our knowledge, this is the first time scRNA-seq has been applied to explore the OT alone and in depth.

Shutdown corner, a large deletion mutant isolated from a haploid mutagenesis screen in zebrafish.

Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened ∼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a ∼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.

A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries.

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries.

Increased Dissociation of Adamantanamines in Influenza A M2 S31N with Partial Block by Rimantadine.

The ubiquitous mutation from serine (WT) to asparagine at residue 31 (S31N) in the influenza A M2 channel renders it insensitive to amantadine (AMT) and rimantadine (RMT) block, but it is unknown whether the inhibition results from weak binding or incomplete block. Two-electrode voltage clamp (TEVC) of transfected Xenopus oocytes revealed that the M2 S31N channel is essentially fully blocked by AMT at 10 mM, demonstrating that, albeit weak, AMT binding in a channel results in complete block of its proton current. In contrast, RMT achieves only a modest degree of block in the M2 S31N channel at 1 mM, with very little increase in block at 10 mM, indicating that the RMT binding site in the channel saturates with only modest block. From exponential curve fits to families of proton current wash-in and wash-out traces, the association rate constant (k1) is somewhat decreased for both AMT and RMT in the S31N, but the dissociation rate constant (k2) is dramatically increased compared with WT. The potentials of mean force (PMF) from adaptive biasing force (ABF) molecular dynamics simulations predict that rate constants should be exquisitely sensitive to the charge state of the His37 selectivity filter of M2. With one exception out of eight cases, predictions from the simulations with one and three charged side chains bracket the experimental rate constants, as expected for the acidic bath used in the TEVC assay. From simulations, the weak binding can be accounted for by changes in the potentials of mean force, but the partial block by RMT remains unexplained.

Lack of skeletal muscle liver kinase B1 alters gene expression, mitochondrial content, inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance.

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice. KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling. KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.

Resistance mechanisms and cross-resistance for a pyridine-pyrimidine amide inhibitor of microtubule polymerization.

Despite their clinical importance, drug resistance remains problematic for microtubule targeting drugs. D4-9-31, a novel microtubule destabilizing agent, has pharmacology that suggests it can overcome common resistance mechanisms and has been shown to remain efficacious in cell and animal models with acquired taxane resistance. To better understand resistance mechanisms and the breadth of cross-resistance with D4-9-31, this study examines the A2780 ovarian cancer cell line as it develops acquired resistance with continuous exposure to D4-9-31. Analyzing cellular responses to D4-9-31 reveals that D4-9-31 resistance is associated with increased mitochondrial respiration, but no cross-resistance to other microtubule targeting agents is observed. Sequencing of transcripts of parental cells and resistant counterparts reveals mutations and altered expression of microtubule-associated genes, but not in genes commonly associated with resistance to microtubule targeting drugs. Additionally, our findings suggest distinct mechanisms drive short- and long-term drug resistance.

Identifying Toxicant-Interacting Genes Using Forward Genetic Screening in Zebrafish.

Forward genetic screening is an extremely powerful method for identifying novel genes driving a broad range of phenotypes. This protocol describes the complete process for conducting a forward genetic screen in zebrafish, including mutagenesis with N-ethyl-N-nitrosourea (ENU), mating, phenotypic screening, and genetic mapping.

SOX10 regulates multiple genes to direct eumelanin versus pheomelanin production in domestic rock pigeon.

The domesticated rock pigeon (Columba livia) has been bred for hundreds of years to display an immense variety of ornamental attributes such as feather color and color patterns. Color is influenced by multiple loci that impact the type and amount of melanin deposited on the feathers. Pigeons homozygous for the "recessive red" mutation, which causes downregulation of Sox10, display brilliant red feathers instead of blue/black feathers. Sox10 encodes a transcription factor important for melanocyte differentiation and function, but the genes that mediate its promotion of black versus red pigment are unknown. Here, we present a transcriptomic comparison of regenerating feathers from wild-type and recessive red pigeons to identify candidate SOX10 targets. Our results identify both known and novel targets, including many genes not previously implicated in pigmentation. These data highlight the value of using novel, emerging model organisms to gain insight into the genetic basis of pigment variation.

Imbalanced sphingolipid signaling is maintained as a core proponent of a cancerous phenotype in spite of metabolic pressure and epigenetic drift.

Tumor heterogeneity may arise through genetic drift and environmentally driven clonal selection for metabolic fitness. This would promote subpopulations derived from single cancer cells that exhibit distinct phenotypes while conserving vital pro-survival pathways. We aimed to identify significant drivers of cell fitness in pancreatic adenocarcinoma (PDAC) creating subclones in different nutrient formulations to encourage differential metabolic reprogramming. The genetic and phenotypic expression profiles of each subclone were analyzed relative to a healthy control cell line (hTert-HPNE). The subclones exhibited distinct variations in protein expression and lipid metabolism. Relative to hTert-HPNE, PSN-1 subclones uniformly maintained modified sphingolipid signaling and specifically retained elevated sphingosine-1-phosphate (S1P) relative to C16 ceramide (C16 Cer) ratios. Each clone utilized a different perturbation to this pathway, but maintained this modified signaling to preserve cancerous phenotypes, such as rapid proliferation and defense against mitochondria-mediated apoptosis. Although the subclones were unique in their sensitivity, inhibition of S1P synthesis significantly reduced the ratio of S1P/C16 Cer, slowed cell proliferation, and enhanced sensitivity to apoptotic signals. This reliance on S1P signaling identifies this pathway as a promising drug-sensitizing target that may be used to eliminate cancerous cells consistently across uniquely reprogrammed PDAC clones.

Development and validation of a pain monitoring app for patients with musculoskeletal conditions (The Keele pain recorder feasibility study).

BACKGROUND: Assessing daily change in pain and related symptoms help in diagnosis, prognosis, and monitoring response to treatment. However, such changes are infrequently assessed, and usually reviewed weeks or months after the start of treatment. We therefore developed a smartphone application (Keele Pain Recorder) to record information on the severity and impact of pain on daily life. Specifically, the study goal was to assess face, content and construct validity of data collection using the Pain Recorder in primary care patients receiving new analgesic prescriptions for musculoskeletal pain, as well as to assess its acceptability and clinical utility. METHODS: The app was developed with Keele's Research User Group (RUG), a clinical advisory group (CAG) and software developer for use on Android devices. The app recorded pain levels, interference, sleep disturbance, analgesic use, mood and side effects. In a feasibility study, patients aged > 18 attending their general practitioner (GP) with a painful musculoskeletal condition were recruited to use the app twice per day for 28 days. Face and construct validity were assessed through baseline and post-study questionnaires (Spearman's rank correlation coefficient). Usability and acceptability were determined through post-study questionnaires, and patient, GP, RUG and CAG interviews. RESULTS: An app was developed which was liked by both patients and GPs. It was felt that it offered the opportunity for GPs to discuss pain control with their patients in a new way. All participants found the app easy to use (it did not interfere with their activities) and results easy to interpret. Strong associations existed between the first 3 days (Spearman r = 0.79) and last 3 days (r = 0.60) of pain levels and intensity scores on the app with the validated questionnaires. CONCLUSIONS: Collaborating with patient representatives and clinical stakeholders, we developed an app which can be used to help clinicians and patients monitor painful musculoskeletal conditions in response to analgesic prescribing. Recordings were accurate and valid, especially, for pain intensity ratings, and it was easy to use. Future work needs to examine how pain trajectories can help manage changes in a patient's condition, ultimately assisting in self-management.