RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Using electronic patient-reported measures to characterize symptoms and improvement in inpatient psychiatric units.

Patient-reported measures are an important tool in personalizing care and monitoring clinical outcomes. This work presents results from the routine collection of self-report measures from individuals (n = 753) admitted to depression and anxiety inpatient units at McLean Hospital. 93.7% participated in the Clinical Measurement Initiative (CMI) between September 2020 and February 2022 on the most established unit. The average time between admission and discharge measures was 12.6 days and an attrition rate of 10.4% was observed on this unit. Missingness of discharge assessments was unrelated to symptom severity or comorbidities. We discuss the feasibility of deploying patient-reported measures as part of routine care in an inpatient psychiatric setting. Systematic evaluation of potential treatment modifiers (e.g., personality disorder, trauma history, and substance misuse) may be valuable in better serving those impacted by psychiatric illness.

State-Level Policy Environments, Discrimination, and Victimization among Sexual and Gender Minority People.

Legislation has been passed in some states to reduce discrimination and victimization toward sexual and gender minority people (SGM; people who are not solely heterosexual and/or whose gender identity is not equal to what is socially associated with sex assigned at birth). The purpose of these analyses is to test whether state-level policy environments are associated with past-year discrimination and victimization among SGM people. Cross-sectional data from The Population Research in Identity and Disparities for Equality (PRIDE) Study annual questionnaire (collected 2018−2019), a national study of the health of SGM adults in the USA, were used for these analyses. Measures included related to discrimination, victimization, and demographic characteristics. State-level policy environments were measured using data from the Movement Advancement Project. Logistic regression analyses evaluated state-level policy environment scores and past-year discrimination and victimization among gender identity categories. In this sample, 7044 people (gender minority n = 2530) were included. Cisgender sexual minority (odds ratio [OR] = 1.007, p = 0.041) and the gender expansive subgroup of gender minority people (OR = 1.010, p = 0.047) in states with more protective policy environments had greater odds of discrimination. The gender expansive subgroup was found to have greater odds of victimization in states with more protective policy environments (OR = 1.003, p < 0.05). There was no relationship between state-level policy environments and victimization among any other study groups. SGM people may experience increased risk for discrimination and victimization despite legislative protections, posing continued risks for poor health outcomes and marginalization. Evaluation of factors (e.g., implementation strategies, systems of accountability) that influence the effectiveness of state-level polices on the reported experiences of discrimination and victimization among SGM people is needed.

Engineering Globin Gene Expression.

Hemoglobinopathies, including sickle cell disease and thalassemia, are among the most common inherited genetic diseases worldwide. Due to the relative ease of isolating and genetically modifying hematopoietic stem and progenitor cells, recent gene editing and gene therapy strategies have progressed to clinical trials with promising outcomes; however, challenges remain and necessitate the continued exploration of new gene engineering and cell transplantation protocols. Current gene engineering strategies aim at reactivating the expression of the fetal γ-globin genes in adult erythroid cells. The γ-globin proteins exhibit anti-sickling properties and can functionally replace adult ß-globin. Here, we describe and compare the current genetic engineering procedures that may develop into safe and efficient therapies for hemoglobinopathies in the near future.

Phase Separation and Transcription Regulation: Are Super-Enhancers and Locus Control Regions Primary Sites of Transcription Complex Assembly?

It is proposed that the multiple enhancer elements associated with locus control regions and super-enhancers recruit RNA polymerase II and efficiently assemble elongation competent transcription complexes that are transferred to target genes by transcription termination and transient looping mechanisms. It is well established that transcription complexes are recruited not only to promoters but also to enhancers, where they generate enhancer RNAs. Transcription at enhancers is unstable and frequently aborted. Furthermore, the Integrator and WD-domain containing protein 82 mediate transcription termination at enhancers. Abortion and termination of transcription at the multiple enhancers of locus control regions and super-enhancers provide a large pool of elongation competent transcription complexes. These are efficiently captured by strong basal promoter elements at target genes during transient looping interactions.

Characterization and functional biology of the soybean aleurone layer.

BACKGROUND: Soybean is a globally important oil seed crop. Both the high protein and oil content of soybean seeds make this crop a lucrative commodity. As in higher eukaryotic species with available genomes, the functional annotation of most of soybean's genes still remains to be investigated. A major hurdle in the functional genomics of soybean is a rapid method to test gene constructs before embarking on stable transformation experiments. RESULTS: In this paper we describe the morphology and composition of the persistent single-cell aleurone layer that derives from the endosperm of developing soybean seeds. Its composition compared to cotyledonary tissue indicates the aleurone layer plays a role in both abiotic and biotic stress. The potential utility as the aleurone layer as a transient expression system in soybean was shown. As a near transparent single-cell layer it can be used as a transient expression system to study transgene expression and inter- and intra-cellular targeting as it is amenable to microscopic techniques. CONCLUSION: The transparent single cell aleurone layer was shown to be compositionally comparable to cotyledonary tissue in soybean with an enrichment in oxidative response proteins and shown to be a potential transient expression platform.

Recent Developments in Insurance Coverage.

This article highlights significant insurance coverage cases from October 1, 2016, to September 30, 2017. This past year, insurance coverage law saw many developments addressing a wide variety of issues. While state and federal courts are frequently divided on their approach to coverage issues, this article attempts to identify regional trends with the aim of assisting practitioners nationwide. The following sections are a review of particularly important decisions in insurance coverage law, including cyber insurance, the efficient proximate cause doctrine, declaratory judgment actions, and recent developments in the application of the pollution exclusion in general liability policies.

BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-α occupancy in the IKKB promoter and inhibits NF-κB activity in antiestrogen-resistant human breast cancer cells.

PURPOSE: We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity ("BRCA1-mimetics"), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 ("NSC35446.HCl"), also inhibited the growth of antiestrogen-resistant LCC9 tumor xenografts. The purpose of this study was to investigate the down-stream effects of NSC35446.HCl and its mechanism of action. METHODS: Here, we studied antiestrogen-resistant (LCC9, T47DCO, MCF-7/RR, LY2), ERα-negative (MDA-MB-231, HCC1806, MDA-MB-468), and antiestrogen-sensitive (MCF-7) cell lines. Techniques utilized include RNA-seq, qRT-PCR, cell growth analysis, cell-cycle analysis, Western blotting, luciferase reporter assays, TUNEL assays, in silico analysis of the IKKB gene, and ChIP assays. RESULTS: SC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen-resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ERα-negative breast cancer cell lines. IKKB (IKKß, IKBKB), an upstream activator of NF-κB, was identified as a BRCA1-mimetic-regulated gene based on an RNA-seq analysis. NSC35446.HCl inhibited IKKB, IKKA, and IKKG/NEMO mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB activity and expression of NF-κB target genes. In silico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ERα was recruited to the ERE-like full-site and five of the nine half-sites and that ERα recruitment was inhibited by NSC35446.HCl in LCC9 and T47DCO cells. CONCLUSIONS: These studies identify functional EREs in the IKKB promoter and identify IKKB as an ERα and NSC35446.HCl-regulated gene, and they suggest that NF-κB and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.

Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF).

Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother's breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N' terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 µg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.

The Potential for Engineering Enhanced Functional-Feed Soybeans for Sustainable Aquaculture Feed.

Aquaculture is the most rapidly growing segment of global animal production that now surpasses wild-capture fisheries production and is continuing to grow 10% annually. Sustainable aquaculture needs to diminish, and progressively eliminate, its dependence on fishmeal-sourced feed from over-harvested fisheries. Sustainable aquafeed sources will need to be primarily of plant-origin. Soybean is currently the primary global vegetable-origin protein source for aquaculture. Direct exchange of soybean meal for fishmeal in aquafeed has resulted in reduced growth rates due in part to soybean's anti-nutritional proteins. To produce soybeans for use in aquaculture feeds a new conventional line has been bred termed Triple Null by stacking null alleles for the feed-relevant proteins Kunitz Trypsin Inhibitor, lectin, and P34 allergen. Triple Null is now being further enhanced as a platform to build additional transgene traits for vaccines, altered protein composition, and to produce high levels of ß-carotene an intrinsic orange-colored aquafeed marker to distinguish the seeds from commodity beans and as the metabolic feedstock precursor of highly valued astaxanthin.

A new class of small molecule estrogen receptor-alpha antagonists that overcome anti-estrogen resistance.

Previous studies indicate that BRCA1 protein binds to estrogen receptor-alpha (ER) and inhibits its activity. Here, we found that BRCA1 over-expression not only inhibits ER activity in anti-estrogen-resistant LCC9 cells but also partially restores their sensitivity to Tamoxifen. To simulate the mechanism of BRCA1 inhibition of ER in the setting of Tamoxifen resistance, we created a three-dimensional model of a BRCA1-binding cavity within the ER/Tamoxifen complex; and we screened a pharmacophore database to identify small molecules that could fit into this cavity. Among the top 40 "hits", six exhibited potent ER inhibitory activity in anti-estrogen-sensitive MCF-7 cells and four of the six exhibited similar activity (IC50 ≤ 1.0 µM) in LCC9 cells. We validated the model by mutation analysis. Two representative compounds (4631-P/1 and 35466-L/1) inhibited ER-dependent cell proliferation in Tamoxifen-resistant cells (LCC9 and LCC2) and partially restored sensitivity to Tamoxifen. The compounds also disrupted the association of BRCA1 with ER. In electrophoretic mobility shift assays, the compounds caused dissociation of ER from a model estrogen response element. Finally, a modified form of compound 35446 (hydrochloride salt) inhibited growth of LCC9 tumor xenografts at non-toxic concentrations. These results identify a novel group of small molecules that can overcome Tamoxifen resistance.

Occupational asphalt is not associated with head and neck cancer.

BACKGROUND: Epidemiologic studies that evaluate the relationship between occupational asphalt exposure and head and neck cancer have had a limited ability to control for known risk factors such as smoking, alcohol and human papillomavirus (HPV). AIMS: To better elucidate this relationship by including known risk factors in a large case-control study of head and neck squamous cell carcinoma (HNSCC) from the greater Boston area. METHODS: We analysed the relationship between occupational asphalt exposure and HNSCC among men in the Greater Boston area of Massachusetts. Analyses were conducted using unconditional multivariable logistic regression, performed with adjustments for age, race, education, smoking, alcohol consumption and HPV serology. RESULTS: There were 753 cases and 913 controls. No associations between HNSCC and occupational asphalt exposure (neither among ever-exposed nor by occupational duration) were observed for exposures in any occupation or those restricted to the construction industry. We also observed no associations in subgroup analyses of never-smokers and ever-smokers. Adjusting for known risk factors further reduced the estimated effect of asphalt exposure on HNSCC risk. CONCLUSIONS: We found no evidence for an association between occupational asphalt exposure and HNSCC. The null findings from this well-controlled analysis could suggest that the risk estimates stemming from occupational cohort studies may be overestimated due to uncontrolled confounding and enhance the literature available for weighing cancer risk from occupational exposure to bitumen.

Transgenic soya bean seeds accumulating ß-carotene exhibit the collateral enhancements of oleate and protein content traits.

Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 µg ß carotene g(-1) dry seed weight with a desirable 12:1 ratio of ß to α. The ß carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated ß-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the mid-maturation ß-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated ß-carotene, oleic acid and protein traits in the ß-carotene soya beans confer a substantial additive nutritional quality to soya beans.

Small-molecule "BRCA1-mimetics" are antagonists of estrogen receptor-α.

CONTEXT: Resistance to conventional antiestrogens is a major cause of treatment failure and, ultimately, death in breast cancer. OBJECTIVE: The objective of the study was to identify small-molecule estrogen receptor (ER)-α antagonists that work differently from tamoxifen and other selective estrogen receptor modulators. DESIGN: Based on in silico screening of a pharmacophore database using a computed model of the BRCA1-ER-α complex (with ER-α liganded to 17ß-estradiol), we identified a candidate group of small-molecule compounds predicted to bind to a BRCA1-binding interface separate from the ligand-binding pocket and the coactivator binding site of ER-α. Among 40 candidate compounds, six inhibited estradiol-stimulated ER-α activity by at least 50% in breast carcinoma cells, with IC50 values ranging between 3 and 50 µM. These ER-α inhibitory compounds were further studied by molecular and cell biological techniques. RESULTS: The compounds strongly inhibited ER-α activity at concentrations that yielded little or no nonspecific toxicity, but they produced only a modest inhibition of progesterone receptor activity. Importantly, the compounds blocked proliferation and inhibited ER-α activity about equally well in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Representative compounds disrupted the interaction of BRCA1 and ER-α in the cultured cells and blocked the interaction of ER-α with the estrogen response element. However, the compounds had no effect on the total cellular ER-α levels. CONCLUSIONS: These findings suggest that we have identified a new class of ER-α antagonists that work differently from conventional antiestrogens (eg, tamoxifen and fulvestrant).

Soybean seed proteome rebalancing.

The soybean seed's protein content and composition are regulated by both genetics and physiology. Overt seed protein content is specified by the genotype's genetic framework and is selectable as a breeding trait. Within the genotype-specified protein content phenotype soybeans have the capacity to rebalance protein composition to create differing proteomes. Soybeans possess a relatively standardized proteome, but mutation or targeted engineering can induce large-scale proteome rebalancing. Proteome rebalancing shows that the output traits of seed content and composition result from two major types of regulation: genotype and post-transcriptional control of the proteome composition. Understanding the underlying mechanisms that specifies the seed proteome can enable engineering new phenotypes for the production of a high-quality plant protein source for food, feed, and industrial proteins.

Targeting the BRCA1/2 tumor suppressors.

The breast cancer susceptibility genes BRCA1 and BRCA2 are classic tumor suppressor genes that exhibit an autosomal dominant pattern of inheritance with high penetrance. BRCA carriers inherit one mutant BRCA allele and one wild-type allele; and the wild-type allele is invariably deleted or mutated within the tumor. These genes function as caretakers in the maintenance of genomic stability, in part, by participating in homology-directed DNA repair (HDR), an error- free mechanism for the repair of double-strand breaks (DSBs). PARP1 (poly (ADP-ribose) polymerase 1) is an enzyme that functions in the base excision repair (BER) pathway, where its ability to post-translationally modify histones and DNA damage response proteins is required for repair of single-strand breaks (SSBs). In 2005, it was observed that knockdown of PARP1 or treatment with a small molecule PARP inhibitor was far more toxic to cells with BRCA1 or BRCA2 mutations than BRCA1/2-competent cells. This observation is an example of "synthetic lethality", a concept whereby two gene mutations combine to cause cell death, when neither mutation alone is lethal. These results spawned the idea to use PARP inhibitors to treat BRCA1/2 mutant cancers. Here, we will review the basic science underlying the discoveries described above, the preclinical research, and the clinical trials designed to exploit the sensitivity of BRCA1/2 mutant tumor cells to PARP inhibitors. We will also describe problems associated with the use of these agents, including development and mechanisms of drug resistance; and we will provide a forward look at new agents and strategies currently under development.

New approaches to radiation protection.

Radioprotectors are compounds that protect against radiation injury when given prior to radiation exposure. Mitigators can protect against radiation injury when given after exposure but before symptoms appear. Radioprotectors and mitigators can potentially improve the outcomes of radiotherapy for cancer treatment by allowing higher doses of radiation and/or reduced damage to normal tissues. Such compounds can also potentially counteract the effects of accidental exposure to radiation or deliberate exposure (e.g., nuclear reactor meltdown, dirty bomb, or nuclear bomb explosion); hence they are called radiation countermeasures. Here, we will review the general principles of radiation injury and protection and describe selected examples of radioprotectors/mitigators ranging from small-molecules to proteins to cell-based treatments. We will emphasize agents that are in more advanced stages of development.