Hemochromatosis drives acute lethal intestinal responses to hyperyersiniabactin-producing Yersinia pseudotuberculosis.

Hemachromatosis (iron-overload) increases host susceptibility to siderophilic bacterial infections that cause serious complications, but the underlying mechanisms remain elusive. The present study demonstrates that oral infection with hyperyersiniabactin (Ybt) producing Yersinia pseudotuberculosis Δfur mutant (termed Δfur) results in severe systemic infection and acute mortality to hemochromatotic mice due to rapid disruption of the intestinal barrier. Transcriptome analysis of Δfur-infected intestine revealed up-regulation in cytokine-cytokine receptor interactions, the complement and coagulation cascade, the NF-κB signaling pathway, and chemokine signaling pathways, and down-regulation in cell-adhesion molecules and Toll-like receptor signaling pathways. Further studies indicate that dysregulated interleukin (IL)-1ß signaling triggered in hemachromatotic mice infected with Δfur damages the intestinal barrier by activation of myosin light-chain kinases (MLCK) and excessive neutrophilia. Inhibiting MLCK activity or depleting neutrophil infiltration reduces barrier disruption, largely ameliorates immunopathology, and substantially rescues hemochromatotic mice from lethal Δfur infection. Moreover, early intervention of IL-1ß overproduction can completely rescue hemochromatotic mice from the lethal infection.

Depletion of NK Cells Improves Cognitive Function in the Alzheimer Disease Mouse Model.

Despite mounting evidence suggesting the involvement of the immune system in regulating brain function, the specific role of immune and inflammatory cells in neurodegenerative diseases remain poorly understood. In this study, we report that depletion of NK cells, a type of innate lymphocytes, alleviates neuroinflammation, stimulates neurogenesis, and improves cognitive function in a triple-transgenic Alzheimer disease (AD) mouse model. NK cells in the brains of triple-transgenic AD mouse model (3xTg-AD) mice exhibited an enhanced proinflammatory profile. Depletion of NK cells by anti-NK1.1 Abs drastically improved cognitive function of 3xTg-AD mice. NK cell depletion did not affect amyloid ß concentrations but enhanced neurogenesis and reduced neuroinflammation. Notably, in 3xTg-AD mice depleted of NK cells, microglia demonstrated a homeostatic-like morphology, decreased proliferative response and reduced expression of neurodestructive proinflammatory cytokines. Together, our results suggest a proinflammatory role for NK cells in 3xTg-AD mice and indicate that targeting NK cells might unlock novel strategies to combat AD.

HuR counteracts miR-330 to promote STAT3 translation during inflammation-induced muscle wasting.

Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the STAT3 (signal transducer and activator of transcription 3) mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the STAT3 mRNA by preventing miR-330 (microRNA 330)-mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the STAT3 mRNA-3'untranslated region (UTR) located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the STAT3-3'UTR negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.

Cis-acting super-enhancer lncRNAs as biomarkers to early-stage breast cancer.

BACKGROUND: Increased breast cancer screening over the past four decades has led to a substantial rise in the diagnosis of ductal carcinoma in situ (DCIS). Although DCIS lesions precede invasive ductal carcinoma (IDC), they do not always transform into cancer. The current standard-of-care for DCIS is an aggressive course of therapy to prevent invasive and metastatic disease resulting in over-diagnosis and over-treatment. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive disease. Recent studies show that super-enhancers, in addition to promoting other gene transcription, are themselves transcribed producing super-enhancer associated long noncoding RNAs (SE-lncRNAs). These SE-lncRNAs can interact with their associated enhancer regions in cis and influence activities and expression of neighboring genes. Furthermore, they represent a novel, untapped group of therapeutic targets. METHODS: With an integrative analysis of enhancer loci with global expression of SE-lncRNAs in the MCF10A progression series, we have identified differentially expressed SE-lncRNAs which can identify mechanisms for DCIS to IDC progression. Furthermore, cross-referencing these SE-lncRNAs with patient samples in the The Cancer Genome Atlas (TCGA) database, we have unveiled 27 clinically relevant SE-lncRNAs that potentially interact with their enhancer to regulate nearby gene expression. To complement SE-lncRNA expression studies, we conducted an unbiased global analysis of super-enhancers that are acquired or lost in progression. RESULTS: Here we designate SE-lncRNAs RP11-379F4.4 and RP11-465B22.8 as potential markers of progression of DCIS to IDC through regulation of the expression of their neighboring genes (RARRES1 and miR-200b, respectively). Moreover, we classified 403 super-enhancer regions in MCF10A normal cells, 627 in AT1, 1053 in DCIS, and 320 in CA1 cells. Comparison analysis of acquired/lost super-enhancer regions with super-enhancer regions classified in 47 ER positive patients, 10 triple negative breast cancer (TNBC) patients, and 11 TNBC cell lines reveal critically acquired pathways including STAT signaling and NF-kB signaling. In contrast, protein folding, and local estrogen production are identified as major pathways lost in progression. CONCLUSION: Collectively, these analyses identify differentially expressed SE-lncRNAs and acquired/lost super-enhancers in progression of breast cancer important for promoting DCIS lesions to IDC.

Group 2 innate lymphoid cells are numerically and functionally deficient in the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: The immune pathways in Alzheimer's disease (AD) remain incompletely understood. Our recent study indicates that tissue-resident group 2 innate lymphoid cells (ILC2) accumulate in the brain barriers of aged mice and that their activation alleviates aging-associated cognitive decline. The regulation and function of ILC2 in AD, however, remain unknown. METHODS: In this study, we examined the numbers and functional capability of ILC2 from the triple transgenic AD mice (3xTg-AD) and control wild-type mice. We investigated the effects of treatment with IL-5, a cytokine produced by ILC2, on the cognitive function of 3xTg-AD mice. RESULTS: We demonstrate that brain-associated ILC2 are numerically and functionally defective in the triple transgenic AD mouse model (3xTg-AD). The numbers of brain-associated ILC2 were greatly reduced in 7-month-old 3xTg-AD mice of both sexes, compared to those in age- and sex-matched control wild-type mice. The remaining ILC2 in 3xTg-AD mice failed to efficiently produce the type 2 cytokine IL-5 but gained the capability to express a number of proinflammatory genes. Administration of IL-5, a cytokine produced by ILC2, transiently improved spatial recognition and learning in 3xTg-AD mice. CONCLUSION: Our results collectively indicate that numerical and functional deficiency of ILC2 might contribute to the cognitive impairment of 3xTg-AD mice.

Long noncoding RNA BHLHE40-AS1 promotes early breast cancer progression through modulating IL-6/STAT3 signaling.

Ductal carcinoma in situ (DCIS) is a nonobligate precursor to invasive breast cancer. Only a small percentage of DCIS cases are predicted to progress; however, there is no method to determine which DCIS lesions will remain innocuous from those that will become invasive disease. Therefore, DCIS is treated aggressively creating a current state of overdiagnosis and overtreatment. There is a critical need to identify functional determinants of progression of DCIS to invasive ductal carcinoma (IDC). Interrogating biopsies from five patients with contiguous DCIS and IDC lesions, we have shown that expression of the long noncoding RNA BHLHE40-AS1 increases with disease progression. BHLHE40-AS1 expression supports DCIS cell proliferation, motility, and invasive potential. Mechanistically, BHLHE40-AS1 modulates interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) activity and a proinflammatory cytokine signature, in part through interaction with interleukin enhancer-binding factor 3. These data suggest that BHLHE40-AS1 supports early breast cancer progression by engaging STAT3 signaling, creating an immune-permissive microenvironment.

Transposon Dysregulation Modulates dWnt4 Signaling to Control Germline Stem Cell Differentiation in Drosophila.

Germline stem cell (GSC) self-renewal and differentiation are required for the sustained production of gametes. GSC differentiation in Drosophila oogenesis requires expression of the histone methyltransferase dSETDB1 by the somatic niche, however its function in this process is unknown. Here, we show that dSETDB1 is required for the expression of a Wnt ligand, Drosophila Wingless type mouse mammary virus integration site number 4 (dWnt4) in the somatic niche. dWnt4 signaling acts on the somatic niche cells to facilitate their encapsulation of the GSC daughter, which serves as a differentiation cue. dSETDB1 is known to repress transposable elements (TEs) to maintain genome integrity. Unexpectedly, we found that independent upregulation of TEs also downregulated dWnt4, leading to GSC differentiation defects. This suggests that dWnt4 expression is sensitive to the presence of TEs. Together our results reveal a chromatin-transposon-Wnt signaling axis that regulates stem cell fate.

Role of miR-203 in estrogen receptor-mediated signaling in the rat uterus and endometrial carcinoma.

The role of microRNAs (miRNA) in estrogen receptor (ER) signaling in the uterus and in endometrial cancer is not well understood. We therefore analyzed miRNA expression in uterine samples from a standard 3-day uterotrophic assay using young female adult rats to identify E2-regulated miRNAs. Microarray analysis identified 47 E2 down-regulated miRNAs including miR-30a, and 25 E2up-regulated miRNAs including miR-672, miR-203, and miR-146b. The strongly E2-upregulated miR-203 was selected for further analysis. miR-203 was deleted in the rat endometrial adenocarcinoma cell line, RUCA-I, using CRISPR/CAS9. Five clones devoid of miR-203 expression were generated. Proliferation was reduced and G2-arrest was observed in all miR-203 deficient RUCA-I clones. Transfection with a miR-203-3p mimic partially rescues this effect. Comparison of mRNA expression in three miR-203 knockout clones to wild type RUCA-I cells reveals 566 miR-203-upregulated and 592 miR-203-downregulated genes. 43 of the genes that are upregulated by miR-203 knockout in vitro are downregulated in the uterus by E2. Of these Acer2, Zbtb20, Ptn, Rcbtb2, Mum1l1, Hmgn3, and Nfat5 possess one or more seed sequence matches in their 3'-UTR that are predicted to be targets of miR-203. These data demonstrate the importance of E2 regulated miRNAs in general, and miR-203 in particular, for E2 regulated gene expression and physiological processes including proliferation and cell migration, in the uterus as well as in the etiology of endometrial carcinomas.

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

Integrin α3ß1 controls mRNA splicing that determines Cox-2 mRNA stability in breast cancer cells.

It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin α3ß1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress α3ß1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in α3ß1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of α3ß1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Moreover, Cox-2 mRNA has reduced stability in α3ß1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies α3ß1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.

Antiproliferative effects of pomegranate extract in MCF-7 breast cancer cells are associated with reduced DNA repair gene expression and induction of double strand breaks.

Pomegranate extract (PE) inhibits the proliferation of breast cancer cells and stimulates apoptosis in MCF-7 breast cancer cells. While PE is a potent antioxidant, the present studies were conducted to examine the mechanisms of action of PE beyond antioxidation by studying cellular and molecular mechanisms underlying breast tumorigenesis. PE inhibited cell growth by inducing cell cycle arrest in G2 /M followed by the induction of apoptosis. In contrast, antioxidants N-acetylcysteine and Trolox did not affect cell growth at doses containing equivalent antioxidant capacity as PE, suggesting that growth inhibition by PE cannot solely be attributed to its high antioxidant potential. DNA microarray analysis revealed that PE downregulated genes associated with mitosis, chromosome organization, RNA processing, DNA replication and DNA repair, and upregulated genes involved in regulation of apoptosis and cell proliferation. Both microarray and quantitative RT-PCR indicated that PE downregulated important genes involved in DNA double strand break (DSB) repair by homologous recombination (HR), such as MRE11, RAD50, NBS1, RAD51, BRCA1, BRCA2, and BRCC3. Downregulation of HR genes correlated with increased levels of their predicted microRNAs (miRNAs), miR-183 (predicted target RAD50) and miR-24 (predicted target BRCA1), suggesting that PE may regulate miRNAs involved in DNA repair processes. Further, PE treatment increased the frequency of DSBs. These data suggest that PE downregulates HR which sensitizes cells to DSBs, growth inhibition and apoptosis. Because HR represents a novel target for cancer therapy, downregulation of HR by PE may be exploited for sensitization of tumors to anticancer drugs.

Substrate activation and conformational dynamics of guanosine 5'-monophosphate synthetase.

Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis.

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Nuclear cytoglobin associates with HMGB2 and regulates DNA damage and genome-wide transcriptional output in the vasculature.

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Inhibition of proprotein convertase SKI-1 blocks transcription of key extracellular matrix genes regulating osteoblastic mineralization.

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone (dec-RRLL-cmk), inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Because SKI-1 is required for activation of SREBP and CREB (cAMP-response element-binding protein)/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5-3.0-fold including Phex, Dmp1, COL1A1, COL11A1, and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1, and fibronectin was reduced by both, whereas COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1-activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19-kDa caspase-3. Conversely, overexpression of SKI-1-activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells and possibly osteocytes depends upon the non-apoptotic actions of SKI-1.

Effects of Anticancer Agent P-bi-TAT on Gene Expression Link the Integrin Thyroid Hormone Receptor to Expression of Stemness and Energy Metabolism Genes in Cancer Cells.

Chemically modified forms of tetraiodothyroacetic acid (tetrac), an L-thyroxine derivative, have been shown to exert their anticancer activity at plasma membrane integrin αvß3 of tumor cells. Via a specific hormone receptor on the integrin, tetrac-based therapeutic agents modulate expression of genes relevant to cancer cell proliferation, survival and energy metabolism. P-bi-TAT, a novel bivalent tetrac-containing synthetic compound has anticancer activity in vitro and in vivo against glioblastoma multiforme (GBM) and other types of human cancers. In the current study, microarray analysis was carried out on a primary culture of human GBM cells exposed to P-bi-TAT (10-6 tetrac equivalent) for 24 h. P-bi-TAT significantly affected expression of a large panel of genes implicated in cancer cell stemness, growth, survival and angiogenesis. Recent interest elsewhere in ATP synthase as a target in GBM cells caused us to focus attention on expression of genes involved in energy metabolism. Significantly downregulated transcripts included multiple energy-metabolism-related genes: electron transport chain genes ATP5A1 (ATP synthase 1), ATP51, ATP5G2, COX6B1 (cytochrome c oxidase subunit 6B1), NDUFA8 (NADH dehydrogenase (ubiquinone) FA8), NDUFV2I and other NDUF genes. The NDUF and ATP genes are also relevant to control of oxidative phosphorylation and transcription. Qualitatively similar actions of P-bi-TAT on expression of subsets of energy-metabolism-linked genes were also detected in established human GBM and pancreatic cancer cell lines. In conclusion, acting at αvß3 integrin, P-bi-TAT caused downregulation in human cancer cells of expression of a large number of genes involved in electron transport and oxidative phosphorylation. These observations suggest that cell surface thyroid hormone receptors on αvß3 regulate expression of genes relevant to tumor cell stemness and energy metabolism.

Effects of 1α,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells.

BACKGROUND: There is evidence from epidemiological and in vitro studies that the biological effects of testosterone (T) on cell cycle and survival are modulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in prostate cancer. To investigate the cross talk between androgen- and vitamin D-mediated intracellular signaling pathways, the individual and combined effects of T and 1,25(OH)2D3 on global gene expression in LNCaP prostate cancer cells were assessed. RESULTS: Stringent statistical analysis identifies a cohort of genes that lack one or both androgen response elements (AREs) or vitamin D response elements (VDREs) in their promoters, which are nevertheless differentially regulated by both steroids (either additively or synergistically). This suggests that mechanisms in addition to VDR- and AR-mediated transcription are responsible for the modulation of gene expression. Microarray analysis shows that fifteen miRNAs are also differentially regulated by 1,25(OH)2D3 and T. Among these miR-22, miR-29ab, miR-134, miR-1207-5p and miR-371-5p are up regulated, while miR-17 and miR-20a, members of the miR-17/92 cluster are down regulated. A number of genes implicated in cell cycle progression, lipid synthesis and accumulation and calcium homeostasis are among the mRNA targets of these miRNAs. Thus, in addition to their well characterized effects on transcription, mediated by either or both cognate nuclear receptors, 1,25(OH)2D3 and T regulate the steady state mRNA levels by modulating miRNA-mediated mRNA degradation, generating attenuation feedback loops that result in global changes in mRNA and protein levels. Changes in genes involved in calcium homeostasis may have specific clinical importance since the second messenger Ca2+ is known to modulate various cellular processes, including cell proliferation, cell death and cell motility, which affects prostate cancer tumor progression and responsiveness to therapy. CONCLUSIONS: These data indicate that these two hormones combine to drive a differentiated phenotype, and reinforce the idea that the age dependent decline in both hormones results in the de-differentiation of prostate tumor cells, which results in increased proliferation, motility and invasion common to aggressive tumors. These studies also reinforce the potential importance of miRNAs in prostate cancer progression and therapeutic outcomes.

Metamorphosis and the regenerative capacity of spinal cord axons in Xenopus laevis.

Throughout the vertebrate subphylum, the regenerative potential of central nervous system axons is greatest in embryonic stages and declines as development progresses. For example, Xenopus laevis can functionally recover from complete transection of the spinal cord as a tadpole but is unable to do so after metamorphosing into a frog. Neurons of the reticular formation and raphe nucleus are among those that regenerate axons most reliably in tadpole and that lose this ability after metamorphosis. To identify molecular factors associated with the success and failure of spinal cord axon regeneration, we pharmacologically manipulated thyroid hormone (TH) levels using methimazole or triiodothyronine, to either keep tadpoles in a permanently larval state or induce precocious metamorphosis, respectively. Following complete spinal cord transection, serotonergic axons crossed the lesion site and tadpole swimming ability was restored when metamorphosis was inhibited, but these events failed to occur when metamorphosis was prematurely induced. Thus, the metamorphic events controlled by TH led directly to the loss of regenerative potential. Microarray analysis identified changes in hindbrain gene expression that accompanied regeneration-permissive and -inhibitory conditions, including many genes in the permissive condition that have been previously associated with axon outgrowth and neuroprotection. These data demonstrate that changes in gene expression occur within regenerating neurons in response to axotomy under regeneration-permissive conditions in which normal development has been suspended, and they identify candidate genes for future studies of how central nervous system axons can successfully regenerate in some vertebrates.

Inhibition of inflammatory gene expression in keratinocytes using a composition containing carnitine, thioctic Acid and saw palmetto extract.

Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, ß-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

Rapid, Affordable, and Scalable SARS-CoV-2 Detection From Saliva.

Here we present an inexpensive, rapid, and robust reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection method that is easily scalable, enabling point-of-care facilities and clinical labs to determine results from patients' saliva directly in 30 minutes for less than $2 per reaction. The method uses a novel combination of widely available reagents that can be prepared in bulk, plated, and frozen and remain stable until samples are received. This innovation dramatically reduces preparation time, enabling high-throughput automation and testing with time to results (including setup) in less than 1 hour for 96 patient samples simultaneously when using a 384-well format. By using a dual reporter (phenol red pH indicator for end-point detection and SYTO-9 fluorescent dye for real time), the assay also provides internal validation of results and redundancy in the event of an instrument malfunction.