EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance.

Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

Cardiac progenitors instruct second heart field fate through Wnts.

The heart develops in a synchronized sequence of proliferation and differentiation of cardiac progenitor cells (CPCs) from two anatomically distinct pools of cells, the first heart field (FHF) and second heart field (SHF). Congenital heart defects arise upon dysregulation of these processes, many of which are restricted to derivatives of the FHF or SHF. Of the conserved set of signaling pathways that regulate development, the Wnt signaling pathway has long been known for its importance in SHF development. The source of such Wnts has remained elusive, though it has been postulated that these Wnts are secreted from ectodermal or endodermal sources. The central question remains unanswered: Where do these Wnts come from? Here, we show that CPCs autoregulate SHF development via Wnt through genetic manipulation of a key Wnt export protein (Wls), scRNA-seq analysis of CPCs, and use of our precardiac organoid system. Through this, we identify dysregulated developmental trajectories of anterior SHF cell fate, leading to a striking single ventricle phenotype in knockout embryos. We then applied our findings to our precardiac organoid model and found that Wnt2 is sufficient to restore SHF cell fate in our model of disrupted endogenous Wnt signaling. In this study, we provide a basis for SHF cell fate decision-proliferation vs. differentiation-autoregulated by CPCs through Wnt.

Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, ß- and γ- TLO gene function.

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, ß- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOß2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloß2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

Human Genetic Determinants of Viral Diseases.

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.

Low-Voltage-Activated CaV3.1 Calcium Channels Shape T Helper Cell Cytokine Profiles.

Activation of T cells is mediated by the engagement of T cell receptors (TCRs) followed by calcium entry via store-operated calcium channels. Here we have shown an additional route for calcium entry into T cells-through the low-voltage-activated T-type CaV3.1 calcium channel. CaV3.1 mediated a substantial current at resting membrane potentials, and its deficiency had no effect on TCR-initiated calcium entry. Mice deficient for CaV3.1 were resistant to the induction of experimental autoimmune encephalomyelitis and had reduced productions of the granulocyte-macrophage colony-stimulating factor (GM-CSF) by central nervous system (CNS)-infiltrating T helper 1 (Th1) and Th17 cells. CaV3.1 deficiency led to decreased secretion of GM-CSF from in vitro polarized Th1 and Th17 cells. Nuclear translocation of the nuclear factor of activated T cell (NFAT) was also reduced in CaV3.1-deficient T cells. These data provide evidence for T-type channels in immune cells and their potential role in shaping the autoimmune response.

Balancing openness with Indigenous data sovereignty: An opportunity to leave no one behind in the journey to sequence all of life.

The field of genomics has benefited greatly from its "openness" approach to data sharing. However, with the increasing volume of sequence information being created and stored and the growing number of international genomics efforts, the equity of openness is under question. The United Nations Convention of Biodiversity aims to develop and adopt a standard policy on access and benefit-sharing for sequence information across signatory parties. This standardization will have profound implications on genomics research, requiring a new definition of open data sharing. The redefinition of openness is not unwarranted, as its limitations have unintentionally introduced barriers of engagement to some, including Indigenous Peoples. This commentary provides an insight into the key challenges of openness faced by the researchers who aspire to protect and conserve global biodiversity, including Indigenous flora and fauna, and presents immediate, practical solutions that, if implemented, will equip the genomics community with both the diversity and inclusivity required to respectfully protect global biodiversity.

Mutation of the Drosophila melanogaster serotonin transporter dSERT impacts sleep, courtship, and feeding behaviors.

The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.

Strain background of Candida albicans interacts with SIR2 to alter phenotypic switching.

The genetic background between strains of a single species and within a single strain lineage can significantly impact the expression of biological traits. This genetic variation may also reshape epigenetic mechanisms of cell identity and environmental responses that are controlled by interconnected transcriptional networks and chromatin-modifying enzymes. Histone deacetylases, including sirtuins, are critical regulators of chromatin state and have been directly implicated in governing the phenotypic transition between the 'sterile' white state and the mating-competent opaque state in Candida albicans, a common fungal commensal and pathogen of humans. Here, we found that a previously ambiguous role for the sirtuin SIR2 in C. albicans phenotypic switching is likely linked to the genetic background of mutant strains produced in the RM lineage of SC5314. SIR2 mutants in a specific lineage of BWP17 displayed increased frequencies of switching to the opaque state compared to the wild-type. Loss of SIR2 in other SC5314-derived backgrounds, including newly constructed BWP17 sir2Δ/Δ mutants, failed to recapitulate the increased white-opaque switching frequencies observed in the original BWP17 sir2Δ/Δ mutant background. Whole-genome sequencing revealed the presence of multiple imbalanced chromosomes and large loss of heterozygosity tracts that likely interact with SIR2 to increase phenotypic switching in this BWP17 sir2Δ/Δ mutant lineage. These genomic changes are not found in other SC5314-derived sir2Δ/Δ mutants that do not display increased opaque cell formation. Thus, complex karyotypes can emerge during strain construction that modify mutant phenotypes and highlight the importance of validating strain background when interpreting phenotypes.

Micelle-Promoted Reductive Amination of DNA-Conjugated Amines for DNA-Encoded Library Synthesis.

DNA-encoded libraries (DELs) have become a leading technology for hit identification in drug discovery projects as large, diverse libraries can be generated. DELs are commonly synthesised via split-and-pool methodology; thus, chemical transformations utilised must be highly efficient, proceeding with high conversions. Reactions performed in DEL synthesis also require a broad substrate scope to produce diverse, drug-like libraries. Many pharmaceutical compounds incorporate multiple C-N bonds, over a quarter of which are synthesised via reductive aminations. However, few on-DNA reductive amination procedures have been developed. Herein is reported the application of the micelle-forming surfactant, TPGS-750-M, to the on-DNA reductive amination of DNA-conjugated amines, yielding highly efficient conversions with a broad range of aldehydes, including medicinally relevant heterocyclic and aliphatic substrates. The procedure is compatible with DNA amplification and sequencing, demonstrating its applicability to DEL synthesis.

Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Opioid analgesic use among patients with kidney disease: a systematic review.

INTRODUCTION: Opioid analgesics are often used to manage moderate to severe pain. A significant proportion of patients taking opioids have compromised kidney function. This systematic review aimed to examine the available evidence on the safety and analgesic effect of opioid use in adults with kidney disease. METHODS: We searched eight electronic databases from inception to 26th January 2023. Published original research articles in English reporting on opioid use and pharmacokinetic data among adults with reduced renal function were included. Article screening, data extraction, and quality assessment were conducted by at least two investigators independently. This review was registered prospectively on PROSPERO (ID: CRD42020159091). RESULTS: There were 32 observational studies included, 14 of which reported on morphine use, three involved fentanyl use, two involved hydromorphone use and 13 articles reported on other opioids including codeine, dihydrocodeine, and buprenorphine. CONCLUSION: There is limited and low-quality evidence to inform the safety and analgesic effect of opioid use in reduced renal function. Morphine remains the opioid for which there is the most evidence available on safety and analgesic effect in the context of renal disease. Greater caution and consideration of potential risks and benefits should be applied when using other opioids. Further high-quality studies examining clinical outcomes associated with the use of different opioids and opioid doses in renal disease are warranted.

Persistent headache without neurologic deficit from a spontaneous vertebral artery dissection.

INTRODUCTION: Non-traumatic headache is a common complaint seen in the emergency department (ED), accounting for 2.3% of ED visits per year in the United States (Munoz-Ceron et al., 2019). When approaching the workup and management of headache, an emergency medicine physician is tasked with generating a deadly differential by means of a thorough history and physical exam to determine the next best steps. CASE: A 21-year-old male presented to the emergency department with a debilitating new-onset headache, preceded by an isolated vertiginous event 3 days prior. He was found to have a normal neurologic examination. A non-contrast CT scan of the head revealed a large hypodensity within the left cerebellum with a subsequent MRA of the brain and neck notable for a left vertebral artery dissection, complicated by an ischemic cerebellar stroke. DISCUSSION: With an estimated incidence of 1-5 per 100,000, vertebral artery dissection is a rare cause of stroke within the general population and carries with it a high degree of morbidity and mortality (Rodallec et al., 2008). Vertebral artery dissection is a result of blood penetrating the intimal wall of the artery to form an intramural hematoma. Diagnosis can be difficult in cases presenting subacutely but a thorough history evaluating for red flags and using simple but highly sensitive exams such as the bedside HINTS exam can increase pretest probability of stroke. Clinical syndromes, red flags, and time from onset of symptoms should guide imaging modalities such as CT, CTA, MRI, and MRA in detection of small ischemic changes, intimal flaps, and luminal thromboses. CONCLUSION: Vertebral artery dissection should remain high on the differential for an emergency medicine physician when history is suggestive of a new onset headache, preceded by vertiginous symptoms. An absence of recent trauma and a normal neurologic examination does not eliminate the diagnosis.

Fgf8 promotes survival of nephron progenitors by regulating BAX/BAK-mediated apoptosis.

Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK/BAX and for subsequent steps involving, as yet, undefined roles in kidney development.

The Fgf8 subfamily (Fgf8, Fgf17 and Fgf18) is required for closure of the embryonic ventral body wall.

The closure of the embryonic ventral body wall in amniotes is an important morphogenetic event and is essential for life. Defects in human ventral wall closure are a major class of birth defect and a significant health burden. Despite this, very little is understood about how the ventral body wall is formed. Here, we show that fibroblast growth factor (FGF) ligands FGF8, FGF17 and FGF18 are essential for this process. Conditional mouse mutants for these genes display subtle migratory defects in the abdominal muscles of the ventral body wall and an enlarged umbilical ring, through which the internal organs are extruded. By refining where and when these genes are required using different Cre lines, we show that Fgf8 and Fgf17 are required in the presomitic mesoderm, whereas Fgf18 is required in the somites. This study identifies complex and multifactorial origins of ventral wall defects and has important implications for understanding their origins during embryonic development.

Lewy body disease as a potential negative outcome modifier of glioblastoma treatment: a case report.

BACKGROUND: Elderly patients with glioblastoma are particularly susceptible to the adverse effects of ionizing radiation to the brain. This population also has an increasing prevalence of dementia in the successive seventh, eighth and nineth decade of life, and dementia with Lewy bodies is characterized by pathologic α-synucleins, proteins that take part in neuronal DNA damage repair. CASE PRESENTATION: We report a 77-year-old man, with a history of coronary artery disease and mild cognitive impairment, who experienced subacute behavioral changes over 3 months with wording-finding difficulty, memory loss, confusion, perseveration, and irritable mood. Neuroimaging studies disclosed a 2.5 × 2.4 × 2.7 cm cystic enhancing mass with central necrosis in the left temporal lobe of the brain. Gross total resection of the tumor revealed IDH-1 wild-type glioblastoma. After treatment with radiation and temozolomide chemotherapy, his cognitive status deteriorated rapidly, and he died from unexpected sudden death 2 months after radiation. Autopsy of his brain revealed (i) tumor cells with atypical nuclei and small lymphocytes, (ii) neuronal cytoplasmic inclusions and Lewy bodies that were positive for α-synuclein in the midbrain, pons, amygdala, putamen and globus pallidus, and (iii) no amyloid plaques and only rare neurofibrillary tangles near the hippocampi. CONCLUSIONS: This patient most likely had pre-clinical limbic subtype of dementia with Lewy bodies prior to his diagnosis of glioblastoma. The radiation and temozolomide that was used to treat his tumor may have accelerated neuronal damage due to induction of DNA breakage when his brain was already compromised by pathologic α-synucleins. α-Synucleinopathy could be a negative outcome modifier in glioblastoma patients.

Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1.

Maternally inherited 15q11-13 chromosomal triplications cause a frequent and highly penetrant type of autism linked to increased gene dosages of UBE3A, which encodes a ubiquitin ligase with transcriptional co-regulatory functions. Here, using in vivo mouse genetics, we show that increasing UBE3A in the nucleus downregulates the glutamatergic synapse organizer Cbln1, which is needed for sociability in mice. Epileptic seizures also repress Cbln1 and are found to expose sociability impairments in mice with asymptomatic increases in UBE3A. This Ube3a-seizure synergy maps to glutamate neurons of the midbrain ventral tegmental area (VTA), where Cbln1 deletions impair sociability and weaken glutamatergic transmission. We provide preclinical evidence that viral-vector-based chemogenetic activation of, or restoration of Cbln1 in, VTA glutamatergic neurons reverses the sociability deficits induced by Ube3a and/or seizures. Our results suggest that gene and seizure interactions in VTA glutamatergic neurons impair sociability by downregulating Cbln1, a key node in the expanding protein interaction network of autism genes.

Whole-genome landscape of pancreatic neuroendocrine tumours.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.

Corrigendum: Whole-genome landscape of pancreatic neuroendocrine tumours.

This corrects the article DOI: 10.1038/nature21063.

Cervical cancer screening among English- and Spanish-speaking Hispanic women in an urban safety net health system, 2015-2020.

BACKGROUND: The Hispanic population is heterogeneous with differences in health behaviors across subgroups by nativity and preferred language. We evaluated cervical cancer screening adherence among English- and Spanish-speaking Hispanic patients receiving care at a safety net health system. METHODS: Electronic health records were used to identify 46,094 women aged 30-65. Up to date (UTD) screening was defined based on date of last Pap test, human papillomavirus (HPV) test, or Pap/HPV co-test. RESULTS: Overall, 81.5% of 31,297 Hispanic women were UTD. English-speaking Hispanic women had a lower prevalence of being UTD when compared to Spanish-speaking Hispanic women (aPR: 0.94, 95% CI: 0.93 - 0.96). Further, those with indigent healthcare plans had a higher prevalence of being UTD when compared to those with private insurance (aPR: 1.10, 95% CI: 1.09 - 1.12), while all other health insurance plans were associated with lower UTD screening when compared to private insurance. CONCLUSIONS: These findings suggest screening differences within the Hispanic population, highlighting the need for disaggregated research assessing heterogeneity within racial/ethnic groups, specifically among Hispanic populations.

Comparison of CG, CKD-EPI[AS] and CKD-EPI[ASR] equations to estimate glomerular filtration rate and predict mortality in treatment naïve people living with HIV in Zimbabwe.

BACKGROUND: Renal impairment in people living with HIV (PWH) in Sub-Saharan Africa is common and associated with increased morbidity and mortality. The ideal equation to estimate glomerular filtration rate (eGFR) in this population remains unclear. That which best predicts clinical risk may be the most appropriate while validation studies are awaited. Here we compare the Cockcroft-Gault (CG), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI[ASR]) and the CKD-EPI equation with the race coefficient removed (CKD-EPI[AS]), in a population of anti-retroviral therapy (ART) naïve PWH in Zimbabwe to assess which equation best predicts mortality. METHODS: A retrospective cohort study of treatment naïve PWH at the Newlands Clinic in Harare, Zimbabwe was completed. The study included all patients commencing ART between 2007 and 2019. Predictors of mortality were assessed by multivariable logistic regression. RESULTS: A total of 2991 patients were followed-up for a median of 4.6 years. The cohort was 62.1% female, with 26.1% of patients having at least one comorbidity. The CG equation identified 21.6% of patients as having renal impairment compared with 17.6% with CKD-EPI[AS] and 9.3% with CKD-EPI[ASR]. There was a mortality rate of 9.1% across the study period. The highest mortality risk was seen in those with renal impairment as determined by the CKD-EPI[ASR] equation for both eGFR < 90 and eGFR < 60 with OR 2.97 (95%CI 1.86-4.76) and OR 10.6 (95%CI 3.15-18.04) respectively. CONCLUSION: In treatment naïve PWH in Zimbabwe, the CKD-EPI[ASR] equation identifies patients at highest risk of mortality when compared to the CKD-EPI[AS] and CG equations.