Immunorecognition of Streptococcus mutans secreted proteins protects against caries by limiting tooth adhesion.

INTRODUCTION: Childhood caries, a prevalent chronic disease, affects 60-90 % of children in industrialized regions, leading to lesions in both primary and permanent teeth. This condition precipitates hospital admissions, emergency room visits, elevated treatment costs, and missed school days, thereby impeding the child's academic engagement and increasing the likelihood of caries into adulthood. Despite multiple identified risk factors, significant interpersonal variability remains unexplained. The immune system generates a unique antibody repertoire, essential for maintaining a balanced and healthy oral microbiome. Streptococcus mutans is a primary contributor to the development of caries. METHODS: Employing mass spectrometry, we investigated the S. mutans proteins targeted by antibodies in children both with and without caries, delineating a fundamental suite of proteins discernible by the immune systems of a majority of individuals. Notably, this suite was enriched with proteins pivotal for bacterial adhesion. To ascertain the physiological implications of these discoveries, we evaluated the efficacy of saliva in thwarting S. mutans adherence to dental surfaces. RESULTS: Antibodies in most children recognized a core set of ten S. mutans proteins, with additional proteins identified in some individuals. There was no significant difference in the proteins identified by children with or without caries, but there was variation in antibody binding intensity to some proteins. Functionally, saliva from caries-free individuals, but not children with caries, was found to hinder the binding of S. mutans to teeth. These findings delineate the S. mutans proteome targeted by the immune system and suggest that the inhibition of bacterial adherence to teeth is a primary mechanism employed by the immune system to maintain oral balance and prevent caries formation. CONCLUSIONS: These findings enhance our knowledge of the immune system's function in oral health maintenance and caries prevention, shedding light on how immunoglobulins interact with S. mutans proteins. CLINICAL SIGNIFICANCE: Targeting S. mutans proteins implicated in bacterial adhesion could be a promising strategy for preventing childhood caries.

AI-driven designed protein epigenomics.

The biological revolutions of computationally designed proteins, induced pluripotent stem cells (iPSCs), and the CRISPR-Cas9 system finally enables modifications that can spur deep understanding of spatial requirement of epigenetic information. This commentary describes the utility of a computationally designed protein, EED Binder (EB), when fused to dCas9 (EBdCas9) for identifying critical sites of PRC2 dependent histone H3K27me3 marks in the chromatin. By using EBdCas9 and gRNA, PRC2 function can be inhibited at specific loci, resulting in precise reduction of EZH2 and H3K27me3 marks, and in some (but not all) locations, activation of the gene and functional outcomes (such as regulation of cell cycle or trophoblast transdifferentiation). Interestingly, a functional TATA box located more than 500bp upstream of a TBX18 TSS was found to be repressed by PRC2, supporting the theory that epigenetic regulators control the repression of transcriptional elements on the promoter region. The EBdCas9 technology may provide a useful tool for controlling gene regulation through epigenomic control.

Prolactinoma: Medical and Surgical Considerations.

Prolactinomas are the most common secretory tumor of the pituitary gland. Clinical symptoms may be due to prolactin oversecretion, localized mass effect, or a combination of both. Although the mainstay of prolactinoma management is medical therapy with dopamine agonists, endoscopic endonasal or transcranial surgery, radiation therapy, or a combination of these is an important treatment option in select cases. This article discusses prolactinoma phenotypes, clinical presentations, and clinically pertinent medical and surgical considerations when managing these tumors.

dCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Amino acid primed mTOR activity is essential for heart regeneration.

Heart disease is the leading cause of death with no method to repair damaged myocardium due to the limited proliferative capacity of adult cardiomyocytes. Curiously, mouse neonates and zebrafish can regenerate their hearts via cardiomyocyte de-differentiation and proliferation. However, a molecular mechanism of why these cardiomyocytes can re-enter cell cycle is poorly understood. Here, we identify a unique metabolic state that primes adult zebrafish and neonatal mouse ventricular cardiomyocytes to proliferate. Zebrafish and neonatal mouse hearts display elevated glutamine levels, predisposing them to amino-acid-driven activation of TOR, and that TOR activation is required for zebrafish cardiomyocyte regeneration in vivo. Through a multi-omics approach with cellular validation we identify metabolic and mitochondrial changes during the first week of regeneration. These data suggest that regeneration of zebrafish myocardium is driven by metabolic remodeling and reveals a unique metabolic regulator, TOR-primed state, in which zebrafish and mammalian cardiomyocytes are regeneration competent.

Author Correction: TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Epigenetic metabolites license stem cell states.

It has become clear during recent years that stem cells undergo metabolic remodeling during their activation process. While these metabolic switches take place in pluripotency as well as adult stem cell populations, the rules that govern the switch are not clear. In this review, we summarize some of the transitions in adult and pluripotent cell types and will propose that the key function in this process is the generation of epigenetic metabolites that govern critical epigenetic modifications, and therefore stem cell states.

UTILITY OF AFIRMA GENE EXPRESSION CLASSIFIER FOR EVALUATION OF INDETERMINATE THYROID NODULES AND CORRELATION WITH ULTRASOUND RISK ASSESSMENT: SINGLE INSTITUTIONAL EXPERIENCE.

Objective: We assessed our experience with Afirma gene expression classifier (GEC) combined with sono-graphic risk assessment, using both the American Thyroid Association (ATA) and the Thyroid Imaging Reporting and Data System (TI-RADS) in evaluating indeterminate thyroid nodules. Methods: We identified 98 patients with 101 nodules who had a second fine needle aspiration biopsy (FNA) between January 1, 2014, and September 30, 2017, and sent to Veracyte for cytopathology and subsequent Afirma GEC testing. A second FNA biopsy was performed if the initial cytopathology was either Bethesda III or IV (n = 94) or nondiagnostic (n = 7). We correlated cytopathology, histopathology, and Afirma GEC results with sonographic risk assessment using both the ATA system and TI-RADS. Results: The mean age of the cohort was 57.4 ± 12.3 years; 84% women and 60% white. Repeat FNA was benign in 51 of 101 nodules, and of the remaining 50 nodules, 18 (36%) were GEC-benign and 32 (64%) GEC-suspicious. Eighteen of the 32 GEC-suspicious nodules underwent surgery with the following results: 7 benign (39%), 1 follicular thyroid carcinoma (6%), 6 follicular variant of papillary thyroid cancer (33%), and 4 noninvasive follicular tumor with papillary-like nuclear features (22%). The malignancy rate among the surgical cohort was 39% (without noninvasive follicular tumor with papillary-like nuclear features [NIFTP]) and 61% (with NIFTP) and about 50% and 20% of this group scored in the high suspicion category by ATA and TR5 by TI-RADS, respectively. Conclusion: Afirma GEC was useful in avoiding surgery in one-third of indeterminate nodules and performed similarly to ATA and TI-RADS. However, the use of echogenicity in scoring may underestimate the risk of malignancy in patients with indeterminate nodules. Abbreviations: ATA = American Thyroid Association; AUS = Atypia of Undetermined Significance; FLUS = Follicular Lesion of Undetermined Significance; FN = follicular neoplasm; FNA = fine needle aspiration; FTC = follicular thyroid cancer; FVPTC = follicular variant of papillary thyroid cancer; GEC = Gene Expression Classifier; ND = nondiagnostic; NIFTP = noninvasive follicular tumor with papillary-like nuclear features; TI-RADS = Thyroid Imaging Reporting and Data System; TR = TI-RADS.

Pituitary Dysfunction Among Men Presenting with Hypogonadism.

PURPOSE OF REVIEW: Hypogonadism is a common endocrine dysfunction. This review focuses on the most up-to-date guideline for evaluation of pituitary function among men presenting with signs and symptoms of hypogonadism. RECENT FINDINGS: The clinician must differentiate between primary (testicular) and secondary (pituitary-hypothalamic or central) hypogonadisms and be aware of adult-onset hypogonadism. If gonadotropins are low or inappropriately normal, the clinician must consider potential reversible causes in the hypothalamus-pituitary axis. Also, it is critical to understand the pitfalls of testosterone testing. When clinically indicated, evaluation of other pituitary hormone functions as well as pituitary magnetic resonance imaging may be necessary. Furthermore, it is essential to recognize that pituitary incidentalomas are common. Patients with microprolactinoma are more likely to present with symptoms of sexual dysfunction while those with macroprolactinoma are more likely to present with symptoms of mass effect. Some functional pituitary tumors respond to drug therapy while other nonfunctional tumors require surgical intervention. It is important for the clinician to understand the proper work-up of the hypogonadal patient with pituitary dysfunction and when necessary to refer to an endocrinologist or a neurosurgeon.

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes.

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal.

To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.

First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

Zinc transport and the inhibition of the L-type calcium channel are two separable functions of ZnT-1.

Traditionally, proteins are considered to perform a single role, be it as an enzyme, a channel, a transporter or as a structural scaffold. However, recent studies have described moonlighting proteins that perform distinct and independent functions; for example, TRPM7 is both an ion channel and a kinase. ZnT-1 is a member of the Carrier Diffusion Facilitator family that is expressed throughout the phylogenetic tree from bacteria to humans. Since its cloning in 1995, ZnT-1 is considered a major extruder of Zn2+ based on its capability to protect cells against zinc toxicity. Recently, we reported that ZnT-1 inhibits the L-type calcium channel (LTCC), a major Zn2+ and Ca2+ entry pathway. Here we show that ZnT-1 is a dual-function protein by demonstrating that its abilities to exchange Zn2+/H+ and to inhibit the LTCC are independent of each other and are mediated by different parts of the protein. Specifically, mutations in the membrane-spanning helices that render ZnT-1 unable to transport zinc do not prevent it from inhibiting the LTCC. Moreover, a fragment consisting of the intracellular ZnT-1 C-terminal, which lacks all ion-transfer segments, inhibits the LTCC as efficiently as wild-type ZnT-1. Our data therefore indicates that ZnT-1 performs two structurally independent functions related to zinc homeostasis.

Polyadenylation and degradation of RNA in the mitochondria.

Mitochondria have their own gene expression machinery and the relative abundance of RNA products in these organelles in animals is mostly dictated by their rate of degradation. The molecular mechanisms regulating the differential accumulation of the transcripts in this organelle remain largely elusive. Here, we summarize the present knowledge of how RNA is degraded in human mitochondria and describe the coexistence of stable poly(A) tails and the nonabundant tails, which have been suggested to play a role in the RNA degradation process.

Identification of LACTB2, a metallo-ß-lactamase protein, as a human mitochondrial endoribonuclease.

Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-ß-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.

Selective modulation of cellular voltage-dependent calcium channels by hyperbaric pressure-a suggested HPNS partial mechanism.

Professional deep sea divers experience motor and cognitive impairment, known as High Pressure Neurological Syndrome (HPNS), when exposed to pressures of 100 msw (1.1 MPa) and above, considered to be the result of synaptic transmission alteration. Previous studies have indicated modulation of presynaptic Ca(2+) currents at high pressure. We directly measured for the first time pressure effects on the currents of voltage dependent Ca(2+) channels (VDCCs) expressed in Xenopus oocytes. Pressure selectivity augmented the current in CaV1.2 and depressed it in CaV3.2 channels. Pressure application also affected the channels' kinetics, such as ƮRise, ƮDecay. Pressure modulation of VDCCs seems to play an important role in generation of HPNS signs and symptoms.

Pressure-selective modulation of NMDA receptor subtypes may reflect 3D structural differences.

Professional deep-water divers exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is associated with CNS hyperexcitability. We have previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. We now report that HP (10.1 MPa) differentially affects eight specific NMDAR subtypes. GluN1(1a or 1b) was co-expressed with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. HP increased ionic currents (measured by two electrode voltage clamps) of one subtype, reduced the current in four others, and did not affect the current in the remaining three. 3D theoretical modeling was aimed at revealing specific receptor domains involved with HP selectivity. In light of the information on the CNS spatial distribution of the different NMDAR subtypes, we conclude that the NMDAR's diverse responses to HP may lead to selective HP effects on different brain regions. These discoveries call for further and more specific investigation of deleterious HP effects and suggest the need for a re-evaluation of deep-diving safety guidelines.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling.

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling.

Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.