First-In-Human Phase I Study of Tinengotinib (TT-00420), a Multiple Kinase Inhibitor, as a Single Agent in Patients With Advanced Solid Tumors.

PURPOSE: This first-in-human phase I dose-escalation study evaluated the safety, pharmacokinetics, and efficacy of tinengotinib (TT-00420), a multi-kinase inhibitor targeting fibroblast growth factor receptors 1-3 (FGFRs 1-3), Janus kinase 1/2, vascular endothelial growth factor receptors, and Aurora A/B, in patients with advanced solid tumors. PATIENTS AND METHODS: Patients received tinengotinib orally daily in 28-day cycles. Dose escalation was guided by Bayesian modeling using escalation with overdose control. The primary objective was to assess dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and dose recommended for dose expansion (DRDE). Secondary objectives included pharmacokinetics and efficacy. RESULTS: Forty-eight patients were enrolled (dose escalation, n = 40; dose expansion, n = 8). MTD was not reached; DRDE was 12 mg daily. DLTs were palmar-plantar erythrodysesthesia syndrome (8 mg, n = 1) and hypertension (15 mg, n = 2). The most common treatment-related adverse event was hypertension (50.0%). In 43 response-evaluable patients, 13 (30.2%) achieved partial response (PR; n = 7) or stable disease (SD) ≥ 24 weeks (n = 6), including 4/11 (36.4%) with FGFR2 mutations/fusions and cholangiocarcinoma (PR n = 3; SD ≥ 24 weeks n = 1), 3/3 (100.0%) with hormone receptor (HR)-positive/HER2-negative breast cancer (PR n = 2; SD ≥ 24 weeks n = 1), 2/5 (40.0%) with triple-negative breast cancer (TNBC; PR n = 1; SD ≥ 24 weeks n = 1), and 1/1 (100.0%) with castrate-resistant prostate cancer (CRPC; PR). Four of 12 patients (33.3%; HR-positive/HER2-negative breast cancer, TNBC, prostate cancer, and cholangiocarcinoma) treated at DRDE had PRs. Tinengotinib's half-life was 28-34 hours. CONCLUSIONS: Tinengotinib was well tolerated with favorable pharmacokinetic characteristics. Preliminary findings indicated potential clinical benefit in FGFR inhibitor-refractory cholangiocarcinoma, HER2-negative breast cancer (including TNBC), and CRPC. Continued evaluation of tinengotinib is warranted in phase II trials.

Pulse Oximetry Screening: Association of State Mandates with Emergency Hospitalizations.

We evaluated the association between implementation of state-mandated pulse oximetry screening (POS) and rates of emergency hospitalizations among infants with Critical Congenital Heart Disease (CCHD) and assessed differences in that association across race/ethnicity. We hypothesized that emergency hospitalizations among infants with CCHD decreased after implementation of mandated POS and that the reduction was larger among racial and ethnic minorities compared to non-Hispanic Whites. We utilized statewide inpatient databases from Arizona, California, Kentucky, New Jersey, New York, and Washington State (2010-2014). A difference-in-differences model with negative binomial regression was used. We identified patients with CCHD whose hospitalizations between three days and three months of life were coded as "emergency" or "urgent" or occurred through the emergency department. Numbers of emergency hospitalizations aggregated by month and state were used as outcomes. The intervention variable was an implementation of state-mandated POS. Difference in association across race/ethnicity was evaluated with interaction terms between the binary variable indicating the mandatory policy period and each race/ethnicity group. The model was adjusted for state-specific variables, such as percent of female infants and percent of private insurance. We identified 9,147 CCHD emergency hospitalizations. Among non-Hispanic Whites, there was a 22% (Confidence Interval [CI] 6%-36%) decline in CCHD emergency hospitalizations after implementation of mandated POS, on average. This decline was 65% less among non-Hispanic Blacks compared to non-Hispanic Whites. Our study detected an attenuated association with decreased number of emergency hospitalizations among Black compared to White infants. Further research is needed to clarify this disparity.

Shared Mechanisms Govern HIV Transcriptional Suppression in Circulating CD103+ and Gut CD4+ T Cells.

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor ß (TGF-ß), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-ß signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103- CD4 T cells from the blood and rectum of HIV-negative (HIV-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103- cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.

Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development.

PURPOSE: To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development. METHODS: A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy). RESULTS: D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧ 40 years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades. CONCLUSIONS: Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

Dual trigger with gonadotropin releasing hormone agonist and human chorionic gonadotropin significantly improves live birth rate for women with diminished ovarian reserve.

BACKGROUND: Diminished ovarian reserve (DOR) remains one of the greatest obstacles affecting the chance of a successful live birth after fertility treatment. The present study was set to investigate whether using a "dual trigger" consisted of human chorionic gonadotropin (hCG) plus gonadotropin releasing hormone agonist (GnRH-a) for final oocyte maturation could improve the IVF cycle outcomes for patients with diminished ovarian reserve. METHODS: A total of 427 completed GnRH-antagonist downregulated IVF cycles with fresh embryo transfer (ET) were included in this retrospective analysis. DOR was defined as antral follicle count ≤5 and serum anti-Müllerian hormone level ≤ 1.1 ng/mL. The control group (n = 130) used a 6500 IU of recombinant hCG for trigger, and the study group (n = 297) used 0.2 mg of triptorelin plus 6500 IU of recombinant hCG for trigger. RESULTS: The dual-trigger group had significantly higher oocyte fertilization rate (73.1% vs. 58.6%), clinical pregnancy rate (33.0% vs. 20.7%) and live birth rate (26.9% vs. 14.5%) when compared to the hCG trigger group. In addition, the abortion rate (17.4% vs. 37.0%) and embryo transfer cancellation rate (6.1% vs. 15.4%) were both significantly lower in the dual trigger group. The primary outcome measure was the live birth rate per oocyte retrieval cycle. Secondary outcome measures were embryo transfer cancellation rate, clinical pregnancy rate, implantation rate, chemical pregnancy rate and abortion rate per oocyte retrieval cycle. CONCLUSIONS: Dual triggering the final oocyte maturation with GnRH-a and standard dose of hCG can significantly improve the live birth rate, clinical pregnancy rate, and fertilization rate in women with diminished ovarian reserve undergoing GnRH antagonist down-regulated IVF-ICSI cycles.

Three-Dimensional Quantitative Color-Coding Analysis of Hepatic Arterial Flow Change during Chemoembolization of Hepatocellular Carcinoma.

PURPOSE: To evaluate feasibility of using three-dimensional (3D) quantitative color-coding analysis (QCA) to quantify substasis endpoints after transcatheter arterial chemoembolization of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This single-institution prospective study included 20 patients with HCC who had undergone segmental or subsegmental transcatheter arterial chemoembolization between December 2015 and March 2017. The chemoembolization endpoint was a sluggish anterograde tumor-feeding arterial flow without residual tumor stains. Contrast medium bolus arrival time (BAT) was used as an indicator of arterial flow. BAT of the proper hepatic artery was obtained as a reference point. BATs of the proximal right lobar artery, proximal left lobar artery, and segmental artery that received embolization were analyzed before and after chemoembolization. Wilcoxon signed rank test was used to evaluate the difference between BATs before and after chemoembolization. RESULTS: BATs before and after chemoembolization of the segmental artery that received embolization were 0.47 seconds (interquartile range [IQR], 0.31-0.70 s) and 1.04 seconds (IQR, 0.78-2.01 s; P < .001), respectively. BATs before and after chemoembolization of the proximal left lobar hepatic artery (0.35 s [IQR, 0.11-0.55] and 0.13 s [IQR, 0.05-0.32], P = .025) and right lobar hepatic artery (0.23 s [IQR, 0.13-0.65] and 0.22 s [IQR, 0.08-0.39], P = .027) exhibited no significant change. CONCLUSIONS: 3D QCA is a feasible method for quantifying sluggish segmental arterial flow after transcatheter arterial chemoembolization in patients with HCC.

N-Arylsulfonyl-α-amino carboxamides are potent and selective inhibitors of the chemokine receptor CCR10 that show efficacy in the murine DNFB model of contact hypersensitivity.

Compound 1 ((4-amino-3,5-dichlorophenyl)-1-(4-methylpiperidin-1-yl)-4-(2-nitroimidazol-1-yl)-1-oxobutane-2-sulfonamido) was discovered to be a 690nM antagonist of human CCR10 Ca2+ flux. Optimization delivered (2R)-4-(2-cyanopyrrol-1-yl)-S-(1H-indol-4-yl)-1-(4-methylpiperidin-1-yl)-1-oxobutane-2-sulfonamido (eut-22) that is 300 fold more potent a CCR10 antagonist than 1 and eliminates potential toxicity, mutagenicity, and drug-drug-interaction liabilities often associated with nitroaryls and anilines. eut-22 is highly selective over other GPCR's, including a number of other chemokine receptors. Finally, eut-22 is efficacious in the murine DNFB model of contact hypersensitivity. The efficacy of this compound provides further evidence for the role of CCR10 in dermatological inflammatory conditions.

Improved survival with MEK inhibition in BRAF-mutated melanoma.

BACKGROUND: Activating mutations in serine-threonine protein kinase B-RAF (BRAF) are found in 50% of patients with advanced melanoma. Selective BRAF-inhibitor therapy improves survival, as compared with chemotherapy, but responses are often short-lived. In previous trials, MEK inhibition appeared to be promising in this population. METHODS: In this phase 3 open-label trial, we randomly assigned 322 patients who had metastatic melanoma with a V600E or V600K BRAF mutation to receive either trametinib, an oral selective MEK inhibitor, or chemotherapy in a 2:1 ratio. Patients received trametinib (2 mg orally) once daily or intravenous dacarbazine (1000 mg per square meter of body-surface area) or paclitaxel (175 mg per square meter) every 3 weeks. Patients in the chemotherapy group who had disease progression were permitted to cross over to receive trametinib. Progression-free survival was the primary end point, and overall survival was a secondary end point. RESULTS: Median progression-free survival was 4.8 months in the trametinib group and 1.5 months in the chemotherapy group (hazard ratio for disease progression or death in the trametinib group, 0.45; 95% confidence interval [CI], 0.33 to 0.63; P<0.001). At 6 months, the rate of overall survival was 81% in the trametinib group and 67% in the chemotherapy group despite crossover (hazard ratio for death, 0.54; 95% CI, 0.32 to 0.92; P=0.01). Rash, diarrhea, and peripheral edema were the most common toxic effects in the trametinib group and were managed with dose interruption and dose reduction; asymptomatic and reversible reduction in the cardiac ejection fraction and ocular toxic effects occurred infrequently. Secondary skin neoplasms were not observed. CONCLUSIONS: Trametinib, as compared with chemotherapy, improved rates of progression-free and overall survival among patients who had metastatic melanoma with a BRAF V600E or V600K mutation. (Funded by GlaxoSmithKline; METRIC ClinicalTrials.gov number, NCT01245062.).

Kawasaki Disease Before and During the COVID-19 Pandemic: A Nationwide Population-based Study in the United States.

BACKGROUND: A decrease in the incidence of Kawasaki disease during the COVID-19 pandemic has been reported globally. Yet, previous US studies utilized patient populations of limited size and geographic scope, leaving a knowledge gap regarding the national trend. Employing a large sample size will increase the generalizability of the results and allow for more detailed analyses. METHODS: The observational study using the 2016-2020 National (Nationwide) Inpatient Sample examined changes in the Kawasaki disease hospitalization rate in pediatric patients during the COVID-19 pandemic. Sensitivity analyses examined hospitalizations between October and December each year, as the code for multisystem inflammatory syndrome in children was implemented in October 2020. RESULTS: In total, 24,505 hospitalizations with Kawasaki disease diagnoses were examined. Hospitalization rates were 65.1 and 53.8 per 1,000,000 pediatric population during the prepandemic and pandemic periods, respectively. Sensitivity analyses showed an overall decrease of 36.1%, with larger decreases for patients 1-4 years old (49.6%), males (40.0%), Asians or Pacific Islanders (57.9%) and patients in the South (47.5%), compared with their counterparts. Associations of the pandemic period with longer lengths of stay and higher daily costs were detected (adjusted mean ratio 1.11; P < 0.01 for length of stay, and adjusted mean ratio 1.33, P < 0.01 for costs). CONCLUSIONS: A decrease in the incidence of Kawasaki disease during the COVID-19 pandemic was observed nationwide in the United States. Contrary to a report from Japan, we did not observe a relationship between population density and a decrease in Kawasaki disease hospitalization. More detailed analyses in targeted geographical areas may provide further insights.

In vitro and in vivo pharmacokinetics, disposition, and drug-drug interaction potential of tinengotinib (TT-00420), a promising investigational drug for treatment of cholangiocarcinoma and other solid tumors.

Early-stage clinical evaluation of tinengotinib (TT-00420) demonstrated encouraging preliminary efficacies in multiple types of refractory cancers, including fibroblast growth factor receptors (FGFR) inhibitors relapsed cholangiocarcinoma (CCA), castrate-resistant prostate cancer (CRPC), and HR+/HER2- breast cancer and triple negative breast cancer (TNBC). To further evaluate drug-like properties of the drug candidate, it is imperative to understand its metabolism and pharmacokinetic properties. This manuscript presented the investigation results of in vitro permeability, plasma protein binding, metabolic stability, metabolite identification, and drug-drug interaction of tinengotinib. Preclinical ADME (absorption, distribution, excretion, and metabolism) studies in rats and dogs was also conducted using a radioactive labeled tinengotinib, [14C]tinengotinib. Tinengotinib was found to have high permeability and high plasma protein binding and equally distributed between blood and plasma. There were no unique metabolites in human liver microsomes and tinengotinib showed moderate hepatic clearance. Tinengotinib is neither a potential inhibitor nor an inducer of P450 enzymes at clinically relevant concentrations, and unlikely to cause drug-drug interactions when used in combination with other drugs mediated by a key transporter, either as victim or perpetrator. Taken together, tinengotinib demonstrated a minimal risk of clinically relevant drug-drug interactions. Tinengotinib showed good oral bioavailability and dose-dependent exposures in both rat and dog after oral administration. The total radioactivity was largely distributed in the gastrointestinal system and liver, and tinengotinib could not easily pass through the blood-brain barrier. The major drug-related component in rat and dog plasma was unchanged drug (>89 %) with primary route of elimination via feces (>93 % of the dose) and minor via renal excretion (<4 % of the dose). Tinengotinib metabolism is mediated largely by CYP3A4, with minor contributions from CYP2D6 and CYP2C8. Major metabolic pathways include oxidation, oxidative cleavage of the morpholine ring, glucuronide and glutathione conjugations. The overall preclinical pharmacokinetics profile supported the selection and development of tinengotinib as a clinical candidate.

Short Interpregnancy Intervals Among Women Experiencing Homelessness in Colorado.

Importance: Short interpregnancy intervals (SIPIs) are associated with increased risk of adverse maternal and neonatal outcomes. Disparities exist across socioeconomic status, but there is little information on SIPIs among women experiencing homelessness. Objective: To investigate (1) differences in rates and characteristics of SIPIs between women experiencing homelessness and domiciled women, (2) whether the association of homelessness with SIPIs differs across races and ethnicities, and (3) whether the association between SIPIs of less than 6 months (very short interpregnancy interval [VSIPIs]) and maternal and neonatal outcomes differs between participant groups. Design, Setting, and Participants: This cohort study used a Colorado statewide database linking the Colorado All Payer Claims Database, Homeless Management Information System, death records, and infant birth records. Participants included all women who gave birth between January 1, 2016, and December 31, 2021. Data were analyzed from September 1, 2022, to May 10, 2023. Exposures: Homelessness and race and ethnicity. Main Outcomes and Measures: The primary outcome consisted of SIPI, a binary variable indicating whether the interval between delivery and conception of the subsequent pregnancy was shorter than 18 months. The association of VSIPI with maternal and neonatal outcomes was also tested. Results: A total of 77 494 women (mean [SD] age, 30.7 [5.3] years) were included in the analyses, of whom 636 (0.8%) were women experiencing homelessness. The mean (SD) age was 29.5 (5.4) years for women experiencing homelessness and 30.7 (5.3) years for domiciled women. In terms of race and ethnicity, 39.3% were Hispanic, 7.3% were non-Hispanic Black, and 48.4% were non-Hispanic White. Associations between homelessness and higher odds of SIPI (adjusted odds ratio [AOR], 1.23 [95% CI, 1.04-1.46]) were found. Smaller associations between homelessness and SIPI were found among non-Hispanic Black (AOR, 0.59 [95% CI, 0.37-0.96]) and non-Hispanic White (AOR, 0.57 [95% CI, 0.39-0.84]) women compared with Hispanic women. A greater association of VSIPI with emergency department visits and low birth weight was found among women experiencing homelessness compared with domiciled women, although no significant differences were detected. Conclusions and Relevance: In this cohort study of women who gave birth from 2016 to 2021, an association between homelessness and higher odds of SIPIs was found. These findings highlight the importance of conception management among women experiencing homelessness. Racial and ethnic disparities should be considered when designing interventions.

Cohesin acetylation promotes sister chromatid cohesion only in association with the replication machinery.

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.

Study on drug substance degradation under headspace GC conditions of residual solvent analysis.

Isobutyraldehyde (IBA) was detected in drug substance (DS) containing an amino acid group using a headspace-gas chromatography (HS-GC) method. High-performance liquid chromatography-mass-spectrometry (HPLC-MS) data from an HS vial confirmed that IBA was a degradant. The HS-GC method was modified to minimize IBA by keeping the HS oven temperature lower than 80 °C.

Tinengotinib (TT-00420), a Novel Spectrum-Selective Small-Molecule Kinase Inhibitor, Is Highly Active Against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous cancer lacking actionable targets. Using a phenotypic screen of TNBC cells, we discovered a novel multiple kinase inhibitor tinengotinib (TT-00420) that strongly inhibited Aurora A/B, FGFR1/2/3, VEGFRs, JAK1/2, and CSF1R in biochemical assays. Exposure to tinengotinib specifically inhibited proliferation across all subtypes of TNBC in vitro and in vivo, while leaving luminal breast cancer cells intact. Incubation of HCC1806 with tinengotinib led to dose-dependent downregulation of genes essential for TNBC cell growth and proliferation. Studies revealed that the potential mechanism of action of tinengotinib involved, predominantly, inhibition of Aurora A or B kinase activity, while inhibition of other pathways contributed to suppression of potency and activity. In vitro treatment of TNBC cell lines or in vivo administration in a syngeneic model with tinengotinib resulted in up-regulation of CXCL10 and 11 or diminished tumor-associated macrophage (TAM) infiltration. Tinengotinib represents a novel combinatorial inhibitory mechanism to treat TNBC. The phase I trial of tinengotinib was completed (ClinicalTrials.gov identifier: NCT03654547).

Sensitivity of Hypocretin System to Chronic Alcohol Exposure: A Human and Animal Study.

Human heroin addicts and mice administered morphine for a 2 week period show a greatly increased number of hypothalamic hypocretin (Hcrt or orexin) producing neurons with a concomitant reduction in Hcrt cell size. Male rats addicted to cocaine similarly show an increased number of detectable Hcrt neurons. These findings led us to hypothesize that humans with alcohol use disorder (AUD) would show similar changes. We now report that humans with AUD have a decreased number and size of detectable Hcrt neurons. In addition, the intermingled melanin concentrating hormone (MCH) neurons are reduced in size. We saw no change in the size and number of tuberomammillary histamine neurons in AUD. Within the Hcrt/MCH neuronal field we found that microglia cell size was increased in AUD brains. In contrast, male rats with 2 week alcohol exposure, sufficient to elicit withdrawal symptoms, show no change in the number or size of Hcrt, MCH and histamine neurons, and no change in the size of microglia. The present study indicates major differences between the response of Hcrt neurons to opioids and that to alcohol in human subjects with a history of substance abuse.

A conserved motif at the C terminus of sororin is required for sister chromatid cohesion.

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.

Single cell genome sequencing of laboratory mouse microbiota improves taxonomic and functional resolution of this model microbial community.

Laboratory mice are widely studied as models of mammalian biology, including the microbiota. However, much of the taxonomic and functional diversity of the mouse gut microbiome is missed in current metagenomic studies, because genome databases have not achieved a balanced representation of the diverse members of this ecosystem. Towards solving this problem, we used flow cytometry and low-coverage sequencing to capture the genomes of 764 single cells from the stool of three laboratory mice. From these, we generated 298 high-coverage microbial genome assemblies, which we annotated for open reading frames and phylogenetic placement. These genomes increase the gene catalog and phylogenetic breadth of the mouse microbiota, adding 135 novel species with the greatest increase in diversity to the Muribaculaceae and Bacteroidaceae families. This new diversity also improves the read mapping rate, taxonomic classifier performance, and gene detection rate of mouse stool metagenomes. The novel microbial functions revealed through our single-cell genomes highlight previously invisible pathways that may be important for life in the murine gastrointestinal tract.

Classification of Activated Microglia by Convolutional Neural Networks.

Microglia are the resident macrophages in the central nervous system. Brain injuries, such as traumatic brain injury, hypoxia, and stroke, can induce inflammatory responses accompanying microglial activation. The morphology of microglia is notably diverse and is one of the prominent manifestations during activation. In this study, we proposed to detect the activated microglia in immunohistochemistry images by convolutional neural networks (CNN). 2D Iba1 images (40µm) were acquired from a control and a cardiac arrest treated Sprague-Dawley rat brain by a scanning microscope using a 20X objective. The training data were a collection of 54,333 single-cell images obtained from the cortex and midbrain areas, and curated by experienced neuroscientists. Results were compared between CNNs with different architectures, including Resnet18, Resnet50, Resnet101, and support vector machine (SVM) classifiers. The highest model performance was found by Resnet18, trained after 120 epochs with a classification accuracy of 95.5%. The findings indicate a potential application for using CNN in quantitative analysis of microglial morphology over regional difference in a large brain section.

Novel protein kinase inhibitor TT-00420 inhibits gallbladder cancer by inhibiting JNK/JUN-mediated signaling pathway.

PURPOSE: This study aimed to investigate the efficiency of our chemically synthesized TT-00420, a novel spectrum-selective multiple protein kinase inhibitor, in cultured cells and animal models of gallbladder cancer (GBC) and explore its potential mechanism. METHODS: Multiple GBC models were established to assess the anti-tumor efficiency, toxicity, and pharmacokinetics of TT-00420. Integrated transcriptomic, proteomic and phosphoproteomic analysis was conducted to identify potential downstream effectors of TT-00420. Western blotting, qRT-PCR, nuclear-cytoplasm separation, and immunofluorescence were performed to confirm the multi-omic results and explore the molecular mechanism of TT-00420. Immunohistochemistry was used to detect FGFR1 and p-FGFR1 expression levels in GBC samples. Autodock software was utilized to investigate the potential binding mode between the TT-00420 and the human FGFR1. RESULTS: We found that TT-00420 exerted potent growth inhibition of GBC cell lines and multiple xenograft models. Treatment of mice with 15 mg/kg TT-00420 via gavage displayed a half-life of 1.8 h in the blood and rapid distribution to the liver, kidneys, lungs, spleen, and tumors at 0.25 h, but no toxicity to these organs over 2 weeks. Multi-omic analysis revealed c-Jun as a potential downstream effector after TT-00420 treatment. Mechanistically, TT-00420 showed rigorous ability to block FGFR1 and its downstream JNK-JUN (S63/S73) signaling pathway, and induce c-Jun S243-dependent MEK/ERK reactivation, leading to FASLG-dependent tumor cell death. Finally, we found that FGFR1 and p-FGFR1 expression was elevated in GBC patients and these levels correlated with decreased patient survival. CONCLUSIONS: TT-00420 shows potent antitumor efficacy and may serve as a novel agent to improve GBC prognosis.