Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility.

Genes are transcribed in a discontinuous pattern referred to as RNA bursting, but the mechanisms regulating this process are unclear. Although many physiological signals, including glucocorticoid hormones, are pulsatile, the effects of transient stimulation on bursting are unknown. Here we characterize RNA synthesis from single-copy glucocorticoid receptor (GR)-regulated transcription sites (TSs) under pulsed (ultradian) and constant hormone stimulation. In contrast to constant stimulation, pulsed stimulation induces restricted bursting centered around the hormonal pulse. Moreover, we demonstrate that transcription factor (TF) nuclear mobility determines burst duration, whereas its bound fraction determines burst frequency. Using 3D tracking of TSs, we directly correlate TF binding and RNA synthesis at a specific promoter. Finally, we uncover a striking co-bursting pattern between TSs located at proximal and distal positions in the nucleus. Together, our data reveal a dynamic interplay between TF mobility and RNA bursting that is responsive to stimuli strength, type, modality, and duration.

Shared Neural Activity But Distinct Neural Dynamics for Cognitive Control in Monkey Prefrontal and Parietal Cortex.

To better understand how prefrontal networks mediate forms of cognitive control disrupted in schizophrenia, we translated a variant of the AX continuous performance task that measures specific deficits in the human disease to 2 male monkeys and recorded neurons in PFC and parietal cortex during task performance. In the task, contextual information instructed by cue stimuli determines the response required to a subsequent probe stimulus. We found parietal neurons encoding the behavioral context instructed by cues that exhibited nearly identical activity to their prefrontal counterparts (Blackman et al., 2016). This neural population switched their preference for stimuli over the course of the trial depending on whether the stimuli signaled the need to engage cognitive control to override a prepotent response. Cues evoked visual responses that appeared in parietal neurons first, whereas population activity encoding contextual information instructed by cues was stronger and more persistent in PFC. Increasing cognitive control demand biased the representation of contextual information toward the PFC and augmented the temporal correlation of task-defined information encoded by neurons in the two areas. Oscillatory dynamics in local field potentials differed between cortical areas and carried as much information about task conditions as spike rates. We found that, at the single-neuron level, patterns of activity evoked by the task were nearly identical between the two cortical areas. Nonetheless, distinct population dynamics in PFC and parietal cortex were evident. suggesting differential contributions to cognitive control.SIGNIFICANCE STATEMENT We recorded neural activity in PFC and parietal cortex of monkeys performing a task that measures cognitive control deficits in schizophrenia. This allowed us to characterize computations performed by neurons in the two areas to support forms of cognitive control disrupted in the disease. Subpopulations of neurons in the two areas exhibited parallel modulations in firing rate; and as a result, all patterns of task-evoked activity were distributed between PFC and parietal cortex. This included the presence in both cortical areas of neurons reflecting proactive and reactive cognitive control dissociated from stimuli or responses in the task. However, differences in the timing, strength, synchrony, and correlation of information encoded by neural activity were evident, indicating differential contributions to cognitive control.

Awareness of having hypertension, diabetes and dyslipidaemia among US adults: The 2011-2018 NHANES data.

AIM: This study aimed to investigate awareness of having hypertension, diabetes and dyslipidaemia and their associated factors among US adults. METHODS: Data from the National Health and Nutrition Examination Survey, including 21,399 adults aged ⩾20 years (pregnant women excluded) collected between 2011 and 2018, were used. Blood pressure was measured using a Baumanometer calibrated mercury true gravity wall model sphygmomanometer. Serum total cholesterol levels were measured using enzymatic assays. The percentage of haemoglobin A1C (HbA1c), which reflects long-term blood glucose levels, was measured and used to identify diabetes. Participants self-reported whether they were told by a doctor that they have hypertension, dyslipidaemia and diabetes. Awareness was defined as alignment between objective and self-reported measures for having the conditions. Sampling weights and the Taylor series linearisation variance estimation method were used in the analyses. RESULTS: The findings showed that 64.06% of people with hypertension, 54.71% of those with dyslipidaemia and 78.40% of those with diabetes were aware of having the respective condition. Age, sex and health insurance were associated with awareness of having all three conditions, but marital status was not associated with any outcome. Weight status was associated with awareness of having hypertension and dyslipidaemia, whereas ethnicity was associated with awareness of having hypertension and diabetes. Relative family income was only associated with awareness of having hypertension. CONCLUSIONS: Large proportions of US adults with hypertension, dyslipidaemia and diabetes are not aware of having the conditions. Interventions targeting groups at higher risk of being unaware of these conditions are needed.

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.

Prophylactic immunization to Helicobacter pylori infection using spore vectored vaccines.

BACKGROUND: Helicobacter pylori infection remains a major public health threat leading to gastrointestinal illness and increased risk of gastric cancer. Mostly affecting populations in developing countries no vaccines are yet available and the disease is controlled by antimicrobials which, in turn, are driving the emergence of AMR. MATERIALS AND METHODS: We have engineered spores of Bacillus subtilis to display putative H. pylori protective antigens, urease subunit A (UreA) and subunit B (UreB) on the spore surface. Following oral dosing of mice with these spores, we evaluated immunity and colonization in animals challenged with H. pylori. RESULTS: Oral immunization with spores expressing either UreA or UreB showed antigen-specific mucosal responses (fecal sIgA) including seroconversion and hyperimmunity. Following challenge, colonization by H. pylori was significantly reduced by up to 1-log. CONCLUSIONS: This study demonstrates the utility of bacterial spores for mucosal vaccination to H. pylori infection. The heat stability and robustness of Bacillus spores coupled with their existing use as probiotics make them an attractive solution for either protection against H. pylori infection or potentially for therapy and control of active infection.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence.

BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Renal denervation reduces atrial remodeling in hypertensive rats with metabolic syndrome.

Atrial fibrillation (AF) is highly prevalent in hypertensive patients with metabolic syndrome and is related to inflammation and activation of the sympathoadrenergic system. The multi-ligand Receptor-for-Advanced-Glycation-End-products (RAGE) activates inflammation-associated tissue remodeling and is regulated by the sympathetic nervous system. Its counterpart, soluble RAGE (sRAGE), serves as anti-inflammatory decoy receptor with protective properties. We investigated the effect of sympathetic modulation by renal denervation (RDN) on atrial remodeling, RAGE/sRAGE and RAGE ligands in metabolic syndrome. RDN was performed in spontaneously hypertensive obese rats (SHRob) with metabolic syndrome compared with lean spontaneously hypertensive rats (SHR) and with normotensive non-obese control rats. Blood pressure and heart rate were measured by telemetry. The animals were killed 12 weeks after RDN. Left atrial (LA) and right atrial (RA) remodeling was assessed by histological analysis and collagen types. Sympathetic innervation was measured by tyrosine hydroxylase staining of atrial nerve fibers, RAGE/sRAGE, RAGE ligands, cytokine expressions and inflammatory infiltrates were analyzed by Western blot and immunofluorescence staining. LA sympathetic nerve fiber density was higher in SHRob (+44%) versus controls and reduced after RDN (-64% versus SHRob). RAGE was increased (+718%) and sRAGE decreased (- 62%) in SHRob as compared with controls. RDN reduced RAGE expression (- 61% versus SHRob), significantly increased sRAGE levels (+162%) and induced a significant decrease in RAGE ligand levels in SHRob (- 57% CML and - 51% HMGB1) with reduced pro-inflammatory NFkB activation (- 96%), IL-6 production (- 55%) and reduced inflammatory infiltrates. This led to a reduction in atrial fibrosis (- 33%), collagen type I content (- 72%), accompanied by reduced LA myocyte hypertrophy (- 21%). Transfection experiments on H9C2 cardiomyoblasts demonstrated that RAGE is directly involved in fibrosis formation by influencing cellular production of collagen type I. In conclusion, suppression of renal sympathetic nerve activity by RDN prevents atrial remodeling in metabolic syndrome by reducing atrial sympathetic innervation and by modulating RAGE/sRAGE balance and reducing pro-inflammatory and pro-fibrotic RAGE ligands, which provides a potential therapeutic mechanism to reduce the development of AF.

Good outcomes after pediatric intraperitoneal kidney transplant.

BACKGROUND: Kidney transplantation in small children is technically challenging. Consideration of whether to use intraperitoneal versus extraperitoneal placement of the graft depends on patient size, clinical history, anatomy, and surgical preference. We report a large single-center experience of intraperitoneal kidney transplantation and their outcomes. METHODS: We conducted a retrospective review of pediatric patients who underwent kidney transplantation from April 2011 to March 2018 at a single large volume center. We identified those with intraperitoneal placement and assessed their outcomes, including graft and patient survival, rejection episodes, and surgical or non-surgical complications. RESULTS: Forty-six of 168 pediatric kidney transplants (27%) were placed intraperitoneally in children mean age 5.5 ± 2.3 years (range 1.6-10 years) with median body weight 18.2 ± 5 kg (range 11.4-28.6 kg) during the study period. Two patients (4%) had vascular complications; 10 (22%) had urologic complications requiring intervention; all retained graft function. Thirteen patients (28%) had prolonged post-operative ileus. Eight (17%) patients had rejection episodes ≤6 months post-transplant. Only one case resulted in graft loss and was associated with recurrent focal segmental glomerular sclerosis (FSGS). Two patients (4%) had chronic rejection and subsequent graft loss by 5-year follow-up. At 7-year follow-up, graft survival was 93% and patient survival was 98%. CONCLUSIONS: The intraperitoneal approach offers access to the great vessels, which allows greater inflow and outflow and more abdominal capacity for an adult donor kidney, which is beneficial in very small patients. Risk of graft failure and surgical complications were not increased when compared to other published data on pediatric kidney transplants.

Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation.

Very little is known about how lipid signaling regulates intima hyperplasia after vascular injury. Herein, we report that deletion and pharmacological inhibition of phospholipase D (PLD)2, which generates the signaling lipid phosphatidic acid (PA), reduced neointimal formation in the mouse carotid artery ligation model. PLD2 deficiency inhibits migration of vascular smooth muscle cells (VSMCs) into the intima in mice as well as migration and formation of membrane ruffles in primary VSMCs. PA specifically binds to the IQ motif-containing guanosine triphosphatase-activating protein 1 (IQGAP1) scaffold protein. The binding between PA and IQGAP is required for the plasma membrane recruitment of IQGAP1. Similar to PLD2 inhibition, knockdown of IQGAP1 blocks ruffle formation and migration in VSMCs, which are rescued by expression of the exogenous IQGAP1 but not the PA binding-deficient mutant. These data reveal that the PLD2-PA-IQGAP1 pathway plays an important role in VSMC migration and injury-induced vascular remodeling, and implicate PLD2 as a candidate target for therapeutic interventions.-Wang, Z., Cai, M., Tay, L. W. R., Zhang, F., Wu, P., Huynh, A., Cao, X., Di Paolo, G., Peng, J., Milewicz, D. M., Du, G. Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation.

Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ).

Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway.

The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects.

1-deamino-8-d-arginine vasopressin (desmopressin [DDAVP]) is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na(+)/Ca(2+) fluxes, before and 2 and 4 hours after DDAVP (0.3 µg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, δ-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of α-granules and GPIIb-IIIa activation induced by adenosine 5'-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na(+)/Ca(2+) concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na(+)/Ca(2+) mobilization. This report indicates that the beneficial hemostatic effect of DDAVP is not limited to an increase in large VWF multimers. An enhancement of platelet procoagulant activity appears to be an additional and (at least in platelet disorders) -possibly clinically relevant mechanism of DDAVP's action.

Monofluorination and Trifluoromethylation of BODIPY Dyes for Prolonged Single-Molecule Detection.

Electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with the Ruppert-Prakesh reagent of dimethyl-, tetramethyl- and pentamethyl-substituted boron dipyrromethenes (BODIPY) are investigated. Monofluorinated dyes are synthesized with low yields (<30%), however trifluoromethyl derivatives are obtained in moderate to high yields (≈40-90%). All compounds are characterized by steady-state and time-resolved fluorescence spectroscopy, the photostability is investigated with fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRF). Monofluorination hardly affects the spectroscopic parameters of the unsubstituted parent compounds, but distinctly enhances the photostability, whereas trifluoromethylation leads to a hypsochromic shift by up to 17 nm in both absorption and emission, slightly enhanced intersystem crossing, and higher photostability. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.

Extracorporeal membrane oxygenation after intravenous injection of paraffin oil.

We present a rare cause of acute respiratory distress syndrome (ARDS) due to an accidental intravascular injection of paraffin oil. While there is no specific therapy, we decided to support the patient with veno-venous extracorporeal membrane oxygenation (V-V ECMO) to allow the ARDS to resolve. A previously healthy 30-year-old man was admitted to the Emergency Department with acute onset respiratory distress following an injection with paraffin oil for cosmetic purposes. In 36 hours, the patient developed severe ARDS and respiratory support with V-V ECMO was initiated. The patient was successfully weaned from ECMO on day 11 and was discharged from hospital in full recovery.

Fragmentation patterns of boron-dipyrromethene (BODIPY) dyes by electrospray ionization high-resolution tandem mass spectrometry.

RATIONALE: 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis. METHODS: The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments. RESULTS: Unusual radical molecular cations ([M](+•)) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M](+•) and [M+H](+) decompositions significantly complicated the interpretation of the MS/MS spectra. CONCLUSIONS: Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS.

Possibility of a field effect transistor based on Dirac particles in semiconducting anatase-TiO2 nanowires.

In Dirac materials, like graphene or topological insulators, massless pseudorelativistic electrons promise new, very fast electronic devices by utilizing the partial suppression of backscattering. However, the semimetal nature of graphene makes the realization of practical field effect transistors difficult, due to small on-off current ratios. Here, we propose a new concept, based on Dirac states inside the conduction (or valence) band of a lightly doped wide band gap semiconductor. With the application of a gate voltage, the Dirac states become populated; that is, the Fermi level is switched between the "classical" high-resistivity semiconducting and the relativistic high-mobility metallic range. We demonstrate by theoretical calculations that such a transition can be realized, for example, in thin anatase nanowires, which have been synthesized before. Ta-doped anatase nanowires offer an excellent possibility to build field effect transistors with high speed and good on-off ratio. Guidelines for finding similar "Dirac semiconductors" are provided.

Expanding Our Knowledge of DICER1 Gene Alterations and Their Role in Thyroid Diseases.

Mutations in DICER1, a gene involved in RNA interference, have been associated with a wide range of multi-organ neoplastic and non-neoplastic conditions. Historically known for its association with pleuropulmonary blastoma, DICER1 syndrome has received more attention due to the association with newly discovered diseases and tumors. Recent studies evaluating DICER1 mutations and DICER1-driven thyroid disease in both pediatric and adult thyroid nodules revealed thyroid disease as the most common manifestation of DICER1 mutations. This study undertakes a comprehensive investigation into DICER1 mutations, focusing on their role in thyroid diseases. Specific attention was given to thyroid follicular nodular disease and differentiated thyroid carcinomas in infancy as highly indicative of germline DICER1 mutation or DICER1 syndrome. Additionally, poorly differentiated thyroid carcinoma and thyroblastoma were identified as potential indicators of somatic DICER1 mutations. Recognizing these manifestations should prompt clinicians to expedite genetic evaluation for this neoplastic syndrome and classify these patients as high risk for additional multi-organ malignancies. This study comprehensively synthesizes the current knowledge surrounding this genetically associated entity, providing intricate details on histologic findings to facilitate its diagnosis.

Promoting Pro-Environmental Behavior in Policymaking: A Text-Mining Approach for Literature Review.

The impact of pro-environmental behavior on policymaking has been an exciting area of research. While the relationship between pro-environmental behavior and policymaking has been explored in numerous studies, there needs to be more synthesis on this topic. This is the first text-mining study of pro-environmental effects in which policymaking is a significant factor. In response, this study, for the first time, takes a novel approach by using text mining in R programming to analyze 30 publications from the Scopus database on pro-environmental behavior in policymaking, highlighting major research themes and prospective research areas for future investigation. Results from text mining yielded 10 topic models, which are presented with a synopsis of the published research and a list of the primary authors, as well as a posterior probability via latent Dirichlet allocation (LDA). Additionally, the study conducts a trend analysis of the top 10 journals with the highest impact factor, considering the influence of each journal's mean citation. The study offers an overview of the impacts of pro-environmental behavior in policymaking, showing the most relevant and frequently discussed themes, introduces the scientific visualization of papers published in the Scopus database, and proposes future study directions. These findings can help researchers and environmental specialists better understand how pro-environmental behavior can be fostered more effectively through policymaking.