SCCA1/SERPINB3 suppresses antitumor immunity and blunts therapy-induced T cell responses via STAT-dependent chemokine production.

Patients with cancer who have high serum levels of squamous cell carcinoma antigen 1 (SCCA1, now referred to as SERPINB3) commonly experience treatment resistance and have a poor prognosis. Despite being a clinical biomarker, the modulation of SERPINB3 in tumor immunity is poorly understood. We found positive correlations of SERPINB3 with CXCL1, CXCL8 (CXCL8/9), S100A8, and S100A9 (S100A8/A9) myeloid cell infiltration through RNA-Seq analysis of human primary cervical tumors. Induction of SERPINB3 resulted in increased CXCL1/8 and S100A8/A9 expression, which promoted monocyte and myeloid-derived suppressor cell (MDSC) migration in vitro. In mouse models, Serpinb3a tumors showed increased MDSC and tumor-associated macrophage (TAM) infiltration, contributing to T cell inhibition, and this was further augmented upon radiation. Intratumoral knockdown (KD) of Serpinb3a resulted in tumor growth inhibition and reduced CXCL1 and S100A8/A expression and MDSC and M2 macrophage infiltration. These changes led to enhanced cytotoxic T cell function and sensitized tumors to radiotherapy (RT). We further revealed that SERPINB3 promoted STAT-dependent expression of chemokines, whereby inhibition of STAT activation by ruxolitinib or siRNA abrogated CXCL1/8 and S100A8/ A9 expression in SERPINB3 cells. Patients with elevated pretreatment SCCA levels and high phosphorylated STAT3 (p-STAT3) had increased intratumoral CD11b+ myeloid cells compared with patients with low SCCA levels and p-STAT3, who had improved overall survival after RT. These findings provide a preclinical rationale for targeting SERPINB3 in tumors to counteract immunosuppression and improve the response to RT.

CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database.

The Comprehensive Antibiotic Resistance Database (CARD; card.mcmaster.ca) combines the Antibiotic Resistance Ontology (ARO) with curated AMR gene (ARG) sequences and resistance-conferring mutations to provide an informatics framework for annotation and interpretation of resistomes. As of version 3.2.4, CARD encompasses 6627 ontology terms, 5010 reference sequences, 1933 mutations, 3004 publications, and 5057 AMR detection models that can be used by the accompanying Resistance Gene Identifier (RGI) software to annotate genomic or metagenomic sequences. Focused curation enhancements since 2020 include expanded ß-lactamase curation, incorporation of likelihood-based AMR mutations for Mycobacterium tuberculosis, addition of disinfectants and antiseptics plus their associated ARGs, and systematic curation of resistance-modifying agents. This expanded curation includes 180 new AMR gene families, 15 new drug classes, 1 new resistance mechanism, and two new ontological relationships: evolutionary_variant_of and is_small_molecule_inhibitor. In silico prediction of resistomes and prevalence statistics of ARGs has been expanded to 377 pathogens, 21,079 chromosomes, 2,662 genomic islands, 41,828 plasmids and 155,606 whole-genome shotgun assemblies, resulting in collation of 322,710 unique ARG allele sequences. New features include the CARD:Live collection of community submitted isolate resistome data and the introduction of standardized 15 character CARD Short Names for ARGs to support machine learning efforts.

Human progranulin-expressing mice as a novel tool for the development of progranulin-modulating therapeutics.

The granulin protein (also known as, and hereafter referred to as, progranulin) is a secreted glycoprotein that contributes to overall brain health. Heterozygous loss-of-function mutations in the gene encoding the progranulin protein (Granulin Precursor, GRN) are a common cause of familial frontotemporal dementia (FTD). Gene therapy approaches that aim to increase progranulin expression from a single wild-type allele, an area of active investigation for the potential treatment of GRN-dependent FTD, will benefit from the availability of a mouse model that expresses a genomic copy of the human GRN gene. Here we report the development and characterization of a novel mouse model that expresses the entire human GRN gene in its native genomic context as a single copy inserted into a defined locus (Hprt) in the mouse genome. We show that human and mouse progranulin are expressed in a similar tissue-specific pattern, suggesting that the two genes are regulated by similar mechanisms. Human progranulin rescues a phenotype characteristic of progranulin-null mice, the exaggerated and early deposition of the aging pigment lipofuscin in the brain, indicating that the two proteins are functionally similar. Longitudinal behavioural and neuropathological analyses revealed no significant differences between wild-type and human progranulin-overexpressing mice up to 18 months of age, providing evidence that long-term increase of progranulin levels is well tolerated in mice. Finally, we demonstrate that human progranulin expression can be increased in the brain using an antisense oligonucleotide that inhibits a known GRN-regulating micro-RNA, demonstrating that the transgene is responsive to potential gene therapy drugs. Human progranulin-expressing mice represent a novel and valuable tool to expedite the development of progranulin-modulating therapeutics.

Redundancy in Kiss1 expression safeguards reproduction in the mouse.

Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1(Cre/+) and Kiss1(Cre/Cre)) have ∼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1(Cre/Cre) mice sire normal-sized litters, female Kiss1(Cre/Cre) mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success.

Dynamic temporal requirement of Wnt1 in midbrain dopamine neuron development.

Wnt1-expressing progenitors generate midbrain dopamine (MbDA) and cerebellum (Cb) neurons in distinct temporal windows and from spatially discrete progenitor domains. It has been shown that Wnt1 and Lmx1a participate in a cross-regulatory loop that is utilized during MbDA neuron development. However, Wnt1 expression dynamically changes over time and precedes that of Lmx1a. The spatial and temporal requirements of Wnt1 in development and specifically its requirement for MbDA neurons remain to be determined. To address these issues, we generated a conditional Wnt1 allele and temporally deleted Wnt1 coupled with genetic lineage analysis. Using this approach, we show that patterning of the midbrain (Mb) and Cb by Wnt1 occurs between the one-somite and the six- to eight-somite stages and is solely dependent on Wnt1 function in the Mb, but not in the Cb. Interestingly, an En1-derived domain persists after the early deletion of Wnt1 and mutant cells express OTX2. However, the En1-derived Wnt1-mutant domain does not contain LMX1a-expressing progenitors, and MbDA neurons are depleted. Thus, we demonstrate an early requirement of Wnt1 for all MbDA neurons. Subsequently, we deleted Wnt1 in the ventral Mb and show a continued late requirement for Wnt1 in MbDA neuron development, but not in LMX1a-expressing progenitors. Specifically, Wnt1 deletion disrupts the birthdating of MbDA neurons and causes a depletion of MbDA neurons positioned medially and a concomitant expansion of MbDA neurons positioned laterally during embryogenesis. Collectively, our analyses resolve the spatial and temporal function of Wnt1 in Mb and Cb patterning and in MbDA neuron development in vivo.

The association between low alcohol use and traffic risk behaviors among Brazilian college students.

Although there are a large number of studies focused on binge drinking and traffic risk behaviors (TRB), little is known regarding low levels of alcohol consumption and its association to TRB. The aim of this cross-sectional study is to examine the association of low to moderate alcohol intake pattern and TRB in college students in Brazil. 7037 students from a National representative sample were selected under rigorous inclusion criteria. All study participants voluntarily fulfilled a structured, anonymous, and self-questionnaire regarding alcohol and drug use, social-demographic data, and TRB. Alcohol was assessed according to the average number of alcoholic units consumed on standard occasions over the past 12 months. The associations between alcohol intake and TRB were summarized with odds ratio and their confidence interval obtained from logistic regression. Compared with abstainers students who consumed only one alcohol unit had the risk of being a passenger in a car driven by a drunk driver increased by almost four times, students who reported using five or more units were increased by almost five times the risk of being involved in a car crash. Compared with students who consumed one alcohol unit, the risk of driving under the influence of alcohol increased four times in students using three alcohol units. Age group, use of illicit drugs, employment status, gender, and marital status significantly influenced occurrence of TRB among college students. Our study highlights the potential detrimental effects of low and moderate pattern of alcohol consumption and its relation to riding with an intoxicated driver and other TRB. These data suggest that targeted interventions should be implemented in order to prevent negative consequences due to alcohol use in this population.

Uncovering novel reproductive defects in neurokinin B receptor null mice: closing the gap between mice and men.

Patients bearing mutations in TAC3 and TACR3 (which encode neurokinin B and its receptor, respectively) have sexual infantilism and infertility due to GnRH deficiency. In contrast, Tacr3(-/-) mice have previously been reported to be fertile. Because of this apparent phenotypic discordance between mice and men bearing disabling mutations in Tacr3/TACR3, Tacr3 null mice were phenotyped with close attention to pubertal development, estrous cyclicity, and fertility. Tacr3(-/-) mice demonstrated normal timing of preputial separation and day of first estrus, markers of sexual maturation. However, at postnatal d 60, Tacr3(-/-) males had significantly smaller testes and lower FSH levels than their wild-type littermates. Tacr3(-/-) females had lower uterine weights and abnormal estrous cyclicity. Approximately half of Tacr3(-/-) females had no detectable corpora lutea on ovarian histology at postnatal d 60. Despite this apparent ovulatory defect, all Tacr3(-/-) females achieved fertility when mated. However, Tacr3(-/-) females were subfertile, having both reduced numbers of litters and pups per litter. The subfertility of these animals was not due to a primary ovarian defect, because they demonstrated a robust response to exogenous gonadotropins. Thus, although capable of fertility, Tacr3-deficient mice have central reproductive defects. The remarkable ability of acyclic female Tacr3 null mice to achieve fertility is reminiscent of the reversal of hypogonadotropic hypogonadism seen in a high proportion of human patients bearing mutations in TACR3. Tacr3 mice are a useful model to examine the mechanisms by which neurokinin B signaling modulates GnRH release.

A unique pair of monozygotic twins with concordant clear cell renal cell carcinoma: a case report.

INTRODUCTION: Genetic predisposition to clear cell renal cell carcinoma (ccRCC) has been linked to disorders such as von Hippel-Lindau (VHL) syndrome. While twin research is a classic approach for elucidating genetic and environmental contributions to disease, no monozygotic twin-pair concordant for ccRCC in the absence of VHL syndrome has been previously reported in the literature or in major twin registries. CLINICAL PICTURE: We describe a unique monozygotic twin-pair concordant for ccRCC, with discordant but early ages of onset of 25 and 38 respectively. Cytogenetic studies and direct sequencing for VHL gene mutations in the second twin proved unremarkable. CONCLUSIONS: This is the fi rst reported case of monozygotic twins concordant for ccRCC in the absence of VHL gene mutation. The early yet discordant, age of onset of disease in both twins suggests both genetic and environmental contributions to ccRCC.

Expression of FOS, EGR-1, and ARC in the amygdala and hippocampus of female rats during formation of the intromission mnemonic of pseudopregnancy.

Pseudopregnancy (PSP) in the female rat is a neuroendocrine condition that is induced by repeated and intermittent vaginocervical stimulation received during mating and involves the expression of bicircadian prolactin surges and cessation of normal estrous cyclicity for 10-12 days postmating. The temporal patterning and number of intromissions received by the female are critical for PSP initiation, and thus, short-term encoding of VCS occurs during transduction of intromissions into PSP. In this experiment, we characterized and compared the mating-induced neural activation patterns within amygdalar and hippocampal regions using expression of the immediate early genes FOS, EGR-1, and ARC. Cycling female rats mated on proestrus received 15 or 5 intromissions under paced or nonpaced mating conditions. High numbers of intromissions during nonpaced mating or low numbers received during paced mating are sufficient to induce PSP, while five nonpaced intromissions and mounts without intromission are insufficient. Here we demonstrate that the CA1 region of the hippocampus was selectively sensitive to PSP-sufficient but not PSP-insufficient mating stimulation by showing significant effects of paced mating for all three IEGs. Paced mating also stimulated the expression of ARC within the basolateral, cortical, and central nuclei of the amygdala. The posterodorsal medial amygdala also showed selective EGR-1 responses to PSP-sufficient mating stimulation. There was no effect of hemisphere on IEG expression. The postmating expression profiles of these IEGs provide evidence that limbic areas involved in encoding and consolidation of memory are involved in initiating the neuroendocrine memory of PSP.