RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients

Purpose

The miniature biodegradable implant siG12D-LODER™ was inserted into a tumor and released a siRNA drug against KRAS(G12D) along four months. This novel siRNA based drug was studied, in combination with chemotherapy, as targeted therapy for Locally Advanced Pancreatic Cancer (LAPC).

Methods

An open-label Phase 1/2a study in the first-line setting of patients with non-operable LAPC was initiated. In this study patients were assigned to receive a single dose of siG12D-LODERs, in three escalating dose cohorts (0.025mg, 0.75mg and 3.0mg). Gemcitabine was given on a weekly basis, following the siG12D-LODER^TM insertion, until disease progression. The recommended dose was further examined with modified FOLFIRINOX. The follow up period was eight weeks and survival until death.

Results

Fifteen patients with LAPC were enrolled. Among the 15 treated patients, the most frequent adverse events observed were grade 1or 2 in severity (89%); five patients experienced serious adverse events (SAEs). In 12 patients analyzed by CT scans, none showed tumor progression, the majority (10/12) demonstrated stable disease and two showed partial response. Decrease in tumor marker CA19-9 was observed in 70% (7/10) of patients. Median overall survival was 15.12 months; 18 month survival was 38.5%.

Conclusions

The combination of siG12D-LODER™ and chemotherapy is well tolerated, safe and demonstrated a potential efficacy in patients with LAPC. {"type":"clinical-trial","attrs":{"text":"NCT01188785","term_id":"NCT01188785"}}NCT01188785     

Neuroanatomical Correlates of Dysglycemia in Young Children With Type 1 Diabetes

Studies of brain structure in type 1 diabetes (T1D) describe widespread neuroanatomical differences related to exposure to glycemic dysregulation in adults and adolescents. In this study, we investigate the neuroanatomical correlates of dysglycemia in very young children with early-onset T1D. Structural magnetic resonance images of the brain were acquired in 142 children with T1D and 68 age-matched control subjects (mean age 7.0 ± 1.7 years) on six identical scanners. Whole-brain volumetric analyses were conducted using voxel-based morphometry to detect regional differences between groups and to investigate correlations between regional brain volumes and measures of glycemic exposure (including data from continuous glucose monitoring). Relative to control subjects, the T1D group displayed decreased gray matter volume (GMV) in bilateral occipital and cerebellar regions (P < 0.001) and increased GMV in the left inferior prefrontal, insula, and temporal pole regions (P = 0.002). Within the T1D group, hyperglycemic exposure was associated with decreased GMV in medial frontal and temporal-occipital regions and increased GMV in lateral prefrontal regions. Cognitive correlations of intelligence quotient to GMV were found in cerebellar-occipital regions and medial prefrontal cortex for control subjects, as expected, but not for the T1D group. Thus, early-onset T1D affects regions of the brain that are associated with typical cognitive development.Glucose dysregulation in type 1 diabetes (T1D) can result in physiological complications, such as neuropathies (1), and has also been linked to an increased risk for cognitive deficits and psychological dysfunction (2,3). Neuroanatomical insult from dysglycemia may be particularly consequential in early childhood, which is a period of dynamic brain development that includes rapid myelination of neurons as well as maturation, modification, and pruning of synapses (4). Although the effects of metabolic perturbations in T1D on central nervous system structure are not fully understood, neuronal damage is generally believed to be a consequence of both hyper- and hypoglycemic states. More specifically, nonenzymatic glycation of neural tissue is believed to lead to increased oxidative stress during hyperglycemia (5), and ultimately neurodegradation, while cell death may be instigated through deficiencies in insulin-sensitive signaling pathways (6). Conversely, energy deprivation during hypoglycemia may abet defective apoptotic processes, spur reactive gliosis, or lead to cellular necrosis via excitotoxicity of glutamate receptors (7,8). Accordingly, an improved understanding of the relation between dysglycemia and brain development, especially in young children, is necessary to better inform medical treatment and ultimately improve clinical outcomes of individuals with T1D.Previous investigations of T1D brain structure have shown increased rates of cerebral atrophy during the course of normal aging (9), with more pronounced effects observed in adults with early-onset diabetes (10). These findings suggest increased vulnerability of the younger brain to the disease. However, differences in total gray matter volume (GMV) and total white matter volume (WMV) relative to control subjects have only been observed in middle-age or older adults (mean age 44 years) (11). More fine-grain analyses using voxel-based morphometry (VBM) in adults with T1D relative to control subjects have detected smaller regional GMV in frontal, temporal, and parieto-occipital regions (12), areas that are responsible for language processing, executive function, and cognition. Neuroanatomical variations in adult T1D also have been linked to severe hypoglycemia exposure, lifetime hemoglobin A_1c (HbA_1c), disease duration, and severity of microangiopathy (12,13).Using cross-sectional and longitudinal analyses in older children and adolescents with T1D (mean age ∼12.6 years), our colleagues at Washington University in St. Louis (14–16) and Stanford University (17) observed significant correlations between neuroanatomical changes in occipital, temporal, frontal, and hippocampal regions and either greater exposure to hyperglycemia (as evidenced by lifetime weighted average HbA_1c) or severe hypoglycemic episodes. However, some of the brain-glycemia associations observed in these studies were contradictory to those reported in adults (12), a phenomenon that may be due to developmentally specific responses to glycemic insults or to the heterogeneity of glycemic exposure in the disease.The current study was designed to determine if T1D in very young children (age range 4.0–10.0 years) is associated with significant changes in GMV or WMV relative to age- and sex-matched nondiabetic control subjects. We also sought to determine if such neuroanatomical variations within the T1D group were correlated with measures of glycemic exposure (e.g., HbA_1c or glucose levels measured with continuous glucose monitors [CGMs]) or with cognitive function. We hypothesized that rapidly developing brain regions are especially vulnerable to deviation from a euglycemic state and that this vulnerability would be reflected in regional differences in GMV and WMV in young children with T1D.     

Critical role for IL-1β in DNA damage-induced mucositis

β-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of β-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1β as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1β is induced by DNA damage via an NF-κB–independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1β receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1β prevents epithelial tight junction dysfunction and alleviates mucositis in β-TrCP–deficient mice. IL-1β antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.Only a few E3 ubiquitin ligases have been studied in vivo by gene disruption for the purpose of elucidating their functions and suitability as potential drug targets (1–3). Beta-transducin repeat containing protein (β-TrCP) is an E3 ubiquitin ligase that affects numerous major cell regulators (1, 4, 5), such as the effector of the Wnt pathway, β-catenin (6); the inhibitor of the NF-κB pathway, IκB (7); the cell cycle regulators CDC25A (8), WEE1 (9), Emi1 (10), and Bora (11); and DNA damage responsive proteins CDC25A and Claspin (12). β-TrCP is encoded by two paralog genes that are thought to be identical in their known biochemical features considering their high degree of sequence homology (77%) and virtually identical substrate recognition “pocket” (13). This redundancy may explain the mild phenotype observed upon in vivo ablation of β-TrCP1; the two best characterized substrates of β-TrCP, β-catenin and IκBα, do not accumulate in β-TrCP1–deficient MEFs (MEF^{β-TrCP1−/−}) (10). Furthermore, residual expression of ∼20% β-TrCP2 activity on a β-TrCP1–null background is sufficient for preserving homeostasis in most tissues (2). To gain a better understanding of in vivo β-TrCP functions, we created a floxed β-TrCP2 allele and crossed it to the β-TrCP1–null mouse (10).Here we show that the primary cellular assault following β-TrCP KO is DNA damage, which, when occurring in the gut, ignites a fatal colitis process that is IL-1β–dependent. This pathological process resembles the one that occurs in human patients following chemotherapy or radiation therapy-induced intestinal mucositis, and will therefore be referred to here as mucositis. We suggest that IL-1β is expressed and secreted by epithelial cells following DNA damage via an unknown mechanism. Expression of IL-1β is NF-κB–independent as observed in different experimental systems following DNA damaging treatments. Our study may have implications for human mucositis; we propose the possibility of using anti–IL-1β treatments [e.g., anakinra, an IL-1 receptor antagonist (IL-1Ra) (14)] as a preventive means for chemoradiation therapy-induced mucositis.     

Embryo classification beyond pregnancy: early prediction of first trimester miscarriage using machine learning

PurposeFirst trimester miscarriage is a major concern in IVF-ET treatments, accounting for one out of nine clinical pregnancies and for up to one out of three recognized pregnancies. To develop a machine learning classifier for predicting the risk of cleavage-stage embryos to undergo first trimester miscarriage based on time-lapse images of preimplantation development.MethodsRetrospective study of a 4-year multi-center cohort of 391 women undergoing intra-cytoplasmatic sperm injection (ICSI) and fresh single or double embryo transfers. The study included embryos with positive indication of clinical implantation based on gestational sac visualization either with first trimester miscarriage or live-birth outcome. Miscarriage was determined based on negative fetal heartbeat indication during the first trimester. Data were recorded and obtained in hospital setting and research was performed in university setting.ResultsA minimal subset of six non-redundant morphodynamic features were screened that maintained high prediction capacity. Features that account for the distribution of the nucleolus precursor bodies within the small pronucleus and pronuclei dynamics were highly predictive of miscarriage outcome as evaluated using the SHapley Additive exPlanations (SHAP) methodology. Using this feature subset, XGBoost and random forest models were trained following a 100-fold Monte-Carlo cross validation scheme. Miscarriage was predicted with AUC 0.68 to 0.69.ConclusionWe report the development of a decision-support tool for identifying the embryos with high risk of miscarriage. Prioritizing embryos for transfer based on their predicted risk of miscarriage in combination with their predicted implantation potential is expected to improve live-birth rates and shorten time-to-pregnancy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10815-022-02619-5.     

Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.Bacteria that live in communities have numerous mechanisms to compete with other strains and species. The ability to acquire nutrients is a major factor dictating the success of a species in a community. In addition, the production of secreted factors, such as bacteriocins, that competitively interfere or antagonize other strains/species, also contributes to a member’s fitness in a community. In the microbe-dense human gut ecosystem, such factors and mechanisms of antagonism by predominant members are just beginning to be described, as are models predicting the relevance of these competitive interactions to the microbial community (1). Bacteroidales is the most abundant order of bacteria in the human colonic microbiota, and also the most temporally stable (2). The fact that numerous gut Bacteroidales species stably cocolonize the human gut at high density raises the question of how these related species and strains interact with each other to promote or limit each other’s growth. We previously showed that coresident Bacteroidales strains intimately interact with each other and exchange large amounts of DNA (3) and also cooperate in the utilization of dietary polysaccharides (4). To date, two types of antagonistic factors/systems have been shown to be produced by human gut Bacteroidales species: secreted antimicrobial proteins (5) and T6SSs (3, 6, 7). However, neither of these antagonistic processes has been analyzed to determine if they provide a competitive advantage in the mammalian intestine.Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems used by some Gram-negative bacteria to intoxicate other bacteria or eukaryotic cells. The T6 apparatus is a multiprotein, cell envelope spanning complex comprised of core Tss proteins. A key component of the machinery is a needle-like structure, similar to the T4 contractile bacteriophage tail, which is assembled in the cytoplasm where it is loaded with toxic effectors (8–10). Contraction of the sheath surrounding the needle apparatus drives expulsion of the needle from the cell, delivering the needle and associated effectors either into the supernatant of in vitro grown bacteria, or across the membrane of prey cells. Identified T6SS effectors include cell wall degrading enzymes (11), proteins that affect cell membranes such as phospholipases (12) and pore-forming toxins (13, 14), proteins that degrade NAD(P)+ (15), and nucleases (16). The effector protein is produced with a cognate immunity protein, typically encoded by the adjacent downstream gene (17), which protects the producing cell from the toxicity of the effector. Although both eukaryotic and bacterial cells are targeted by T6SS effectors (18), most described T6SSs target Gram-negative bacteria.We previously performed a comprehensive analysis of all sequenced human gut Bacteroidales stains and found that more than half contain T6SS loci (7). These T6SSs are similar to the well-described T6SSs of Proteobacteria in that remote orthologs of many Proteobacterial Tss proteins are encoded by Bacteroidales T6SS regions, with the exception of proteins that likely comprise the transmembrane complex, which are distinct. The T6SS loci of human gut Bacteroidales species segregate into three distinct genetic architectures (GA), designated GA1, GA2, and GA3, each with highly identical segments within a GA comprising the core tss genes (7). GA1 and GA2 T6SS loci are present on large ∼80- to 120-kb integrative conjugative elements (ICE) that are extremely similar at the DNA level within a GA. Due to the ability of these T6SS regions to be transferred between strains via ICE, GA1 and GA2 T6SS loci are present in diverse human gut Bacteroidales species. GA3 T6SS loci are confined to Bacteroides fragilis and are not contained on conserved ICE (7).Although T6SS loci of a particular GA are highly identical to each other, each GA has internal regions of variability where the genes differ between strains (7). The variable regions of GA1 and GA2 T6SS loci contain genes encoding the identifiable toxic effector and cognate immunity proteins found in these regions. Unlike the GA1 and GA2 T6SS loci, there are no identifiable genes encoding toxin or immunity proteins in the two variable regions or other areas of GA3 T6SS loci. The present study was designed to answer three fundamental questions regarding GA3 T6SS loci: (i) Because no known effectors/immunity proteins are encoded by these regions, are they involved in bacterial antagonism? And if so, what prey cells do they target? (ii) Do the variable regions contain genes encoding effector and immunity proteins? and (iii) If GA3 T6SSs mediate bacterial antagonism, do they provide a competitive advantage in the mammalian gut?     

Quantification of pericardial effusions by echocardiography and computed tomography

Echocardiography is a well-accepted tool for the diagnosis and quantification of pericardial effusion (PEff). Given the increasing use of computed tomographic (CT) scanning, more PEffs are being initially diagnosed by computed tomography. No study has compared quantification of PEff by computed tomography and echocardiography. The objective of this study was to assess the accuracy of quantification of PEff by 2-dimensional echocardiography and computed tomography compared to the amount of pericardial fluid drained at pericardiocentesis. We retrospectively reviewed an institutional database to identify patients who underwent chest computed tomography and echocardiography before percutaneous pericardiocentesis with documentation of the amount of fluid withdrawn. Digital 2-dimensional echocardiographic and CT images were retrieved and quantification of PEff volume was performed by applying the formula for the volume of a prolate ellipse, π × 4/3 × maximal long-axis dimension/2 × maximal transverse dimension/2 × maximal anteroposterior dimension/2, to the pericardial sac and to the heart. Nineteen patients meeting study qualifications were entered into the study. The amount of PEff drained was 200 to 1,700 ml (mean 674 ± 340). Echocardiographically calculated pericardial effusion volume correlated relatively well with PEff volume (r = 0.73, p <0.001, mean difference -41 ± 225 ml). There was only moderate correlation between CT volume quantification and actual volume drained (r = 0.4, p = 0.004, mean difference 158 ± 379 ml). In conclusion, echocardiography appears a more accurate imaging technique than computed tomography in quantitative assessment of nonloculated PEffs and should continue to be the primary imaging in these patients.     

The representation of lexical-syntactic information: evidence from syntactic and lexical retrieval impairments in aphasia

This study explored lexical-syntactic information - syntactic information that is stored in the lexicon - and its relation to syntactic and lexical impairments in aphasia. We focused on two types of lexical-syntactic information: predicate argument structure (PAS) of verbs (the number and types of arguments the verb selects) and grammatical gender of nouns. The participants were 17 Hebrew-speaking individuals with aphasia who had a syntactic deficit (agrammatism) or a lexical retrieval deficit (anomia) located at the semantic lexicon, the phonological output lexicon, or the phonological output buffer. After testing the participants' syntactic and lexical retrieval abilities and establishing the functional loci of their deficits, we assessed their PAS and grammatical gender knowledge. This assessment included sentence completion, sentence production, sentence repetition, and grammaticality judgment tasks. The participants' performance on these tests yielded several important dissociations. Three agrammatic participants had impaired syntax but unimpaired PAS knowledge. Three agrammatic participants had impaired syntax but unimpaired grammatical gender knowledge. This indicates that lexical-syntactic information is represented separately from syntax, and can be spared even when syntax at the sentence level, such as embedding and movement are impaired. All 5 individuals with phonological output buffer impairment and all 3 individuals with phonological output lexicon impairment had preserved lexical-syntactic knowledge. These selective impairments indicate that lexical-syntactic information is represented at a lexical stage prior to the phonological lexicon and the phonological buffer. Three participants with impaired PAS (aPASia) and impaired grammatical gender who showed intact lexical-semantic knowledge indicate that the lexical-syntactic information is represented separately from the semantic lexicon. This led us to conclude that lexical-syntactic information is stored in a separate syntactic lexicon. A double dissociation between PAS and grammatical gender impairments indicated that different types of lexical-syntactic information are represented separately in this syntactic lexicon.