Bisphosphonate inhibits the expression of cyclin A2 at the transcriptional level in normal human oral keratinocytes.

Nitrogen-containing bisphosphonates (N-BPs) are the most widely used anti-resorptive agents in the treatment of bone-related diseases. N-BPs inhibit bone resorption by specifically targeting osteoclasts, bone-resorbing cells. However, soft tissue toxicity, such as oral or gastrointestinal (GI) ulcerations has frequently been reported in N-BP users, suggesting that N-BPs may also directly target cells other than osteoclasts. Previously, we reported that BPs inhibit proliferation without inducing the apoptosis of normal human oral keratinocytes (NHOKs). However, the molecular mechanisms through which N-BPs inhibit the proliferation of NHOKs are not yet fully understood. In this study, we performed gene expression profiling in N-BP-treated NHOKs and identified cyclin A2 as one of the most commonly downregulated genes. When the NHOKs were treated with N-BPs, we found that the level of cyclin A2 was suppressed in a dose- and time-dependent manner. In addition, the protein level of cyclin A2 was also significantly lower in oral epithelial cells in N-BP-treated oral mucosal tissue constructs. Cyclin A2 promoter reporter assay revealed that N-BPs inhibited the luciferase activity, indicating that the inhibition of cyclin A2 expression occurs at the transcriptional level. Furthermore, N-BPs did not alter the expression of cyclin A2 in normal human oral fibroblasts (NHOFs), suggesting that the effect of N-BPs on cyclin A2 expression may be cell-type specific. Thus, the findings of our study demonstrate that the inhibition of NHOK proliferation by N-BPs is mediated, at least in part, by the suppression of cyclin A2 expression at the transcriptional level, which may explain the underlying mechanisms of soft tissue toxicity by N-BPs

Evaluation of 4 Outcomes Measures in Microtia Treatment: Exposures, Infections, Aesthetics, and Psychosocial Ramifications.

BackgroundIn craniofacial microsomia, microtia and canal atresia pose formidable reconstructive challenges. We review our institutional experience in treating microtia and atresia to identify variables associated with 4 outcomes measures: complications, surgical revisions, aesthetic outcomes, and psychosocial function.MethodsCraniofacial microsomia patients treated at the University of California Los Angeles Craniofacial Clinic between 2008 and 2014 greater than 13 years of age (n = 68) were reviewed for microtia and atresia treatment and outcomes.ResultsIn total, 91.2% of patients diagnosed with craniofacial microsomia presented with microtia, affecting 75 ears. Both a male and right-sided predominance were observed. Fifty-six patients (90.3%) underwent autologous external ear reconstruction at an average age of 8.5 years. Age, type of incision, and size of cartilage framework did not predict total number of surgeries or complications. Severity of ear anomalies correlated with increased number of surgeries (P < 0.001) and decreased aesthetic outcomes (P < 0.001) but not complications. In total, 87.1% of patients with microtia had documented hearing loss, of which the majority were conductive and 18.5% were mixed sensorineural and conductive. Hearing deficits were addressed in 70.4% of patients with external hearing aids, bone anchored hearing aids, or canaloplasty. Of all variables, improvement of psychosocial function was correlated only to hearing loss treatment of any type (P = 0.01).ConclusionsOn evaluation of surgical and patient characteristics, severity of microtia predicted the total number of surgical revisions performed and aesthetic ratings. In addition, we found that the only factor that correlated with improved patient and parent-reported psychosocial outcomes was treatment of hearing loss

A New Approach toward Transition State Spectroscopy

Chirped-Pulse millimetre-Wave (CPmmW) rotational spectroscopy provides a new class of information about photolysis transition state(s). Measured intensities in rotational spectra determine species-isomer-vibrational populations, provided that rotational populations can be thermalized. The formation and detection of S0 vinylidene is discussed in the limits of low and high initial rotational excitation. CPmmW spectra of 193 nm photolysis of Vinyl Cyanide (Acrylonitrile) contain J=0-1 transitions in more than 20 vibrational levels of HCN, HNC, but no transitions in vinylidene or highly excited local-bender vibrational levels of acetylene. Reasons for the non-observation of the vinylidene co-product of HCN are discussed.Comment: Accepted by Faraday Discussion

The biological embedding of early-life socioeconomic status and family adversity in children's genome-wide DNA methylation.

AimTo examine variation in child DNA methylation to assess its potential as a pathway for effects of childhood social adversity on health across the life course.Materials & methodsIn a diverse, prospective community sample of 178 kindergarten children, associations between three types of social experience and DNA methylation within buccal epithelial cells later in childhood were examined.ResultsFamily income, parental education and family psychosocial adversity each associated with increased or decreased DNA methylation (488, 354 and 102 sites, respectively) within a unique set of genomic CpG sites. Gene ontology analyses pointed to genes serving immune and developmental regulation functions.ConclusionFindings provided support for DNA methylation as a biomarker linking early-life social experiences with later life health in humans

In an in vitro model of human tuberculosis, monocyte-microglial networks regulate matrix metalloproteinase-1 and -3 gene expression and secretion via a p38 mitogen activated protein kinase-dependent pathway.

BACKGROUND: Tuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. However, relatively little is known about the regulation of these MMPs in the CNS. METHODS: Using a cellular model of CNS TB, we stimulated a human microglial cell line (CHME3) with conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb). MMP-1 and MMP-3 secretion was detected using ELISAs confirmed with casein zymography or western blotting. Key results of a phospho-array profile that detects a wide range of kinase activity were confirmed with phospho-Western blotting. Chemical inhibition (SB203580) of microglial cells allowed investigation of expression and secretion of MMP-1 and MMP-3. Finally we used promoter reporter assays employing full length and MMP-3 promoter deletion constructs. Student's t-test was used for comparison of continuous variables and multiple intervention experiments were compared by one-way ANOVA with Tukey's correction for multiple pairwise comparisons. RESULTS: CoMTb up-regulated microglial MMP-1 and MMP-3 secretion in a dose- and time-dependent manner. The phospho-array profiling showed that the major increase in kinase activity due to CoMTb stimulation was in p38 mitogen activated protein kinase (MAPK), principally the α and γ subunits. p38 phosphorylation was detected at 15 minutes, with a second peak of activity at 120 minutes. High basal extracellular signal-regulated kinase activity was further increased by CoMTb. Secretion and expression of MMP-1 and MMP-3 were both p38 dependent. CoMTb stimulation of full length and MMP-3 promoter deletion constructs demonstrated up-regulation of activity in the wild type but a suppression site between -2183 and -1612 bp. CONCLUSIONS: Monocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 MAPK in tuberculosis. p38 is therefore a potential target for adjuvant therapy in CNS TB

BRIM-P: A phase I, open-label, multicenter, dose-escalation study of vemurafenib in pediatric patients with surgically incurable, BRAF mutation-positive melanoma.

BACKGROUND: Vemurafenib, a selective inhibitor of BRAF kinase, is approved for the treatment of adult stage IIIc/IV BRAF V600 mutation-positive melanoma. We conducted a phase I, open-label, dose-escalation study in pediatric patients aged 12-17 years with this tumor type (NCT01519323). PROCEDURE: Patients received vemurafenib orally until disease progression. Dose escalation was conducted using a 3 + 3 design. Patients were monitored for dose-limiting toxicities (DLTs) during the first 28 days of treatment to determine the maximum tolerated dose (MTD). Safety/tolerability, tumor response, and pharmacokinetics were evaluated. RESULTS: Six patients were enrolled (720 mg twice daily [BID], n = 3; 960 mg BID [n = 3]). The study was terminated prematurely due to low enrollment. No DLTs were observed; thus, the MTD could not be determined. All patients experienced at least one adverse event (AE); the most common were diarrhea, headache, photosensitivity, rash, nausea, and fatigue. Three patients experienced serious AEs, one patient developed secondary cutaneous malignancies, and five patients died following disease progression. Mean steady-state plasma concentrations of vemurafenib following 720 mg and 960 mg BID dosing were similar or higher, respectively, than in adults. There were no objective responses. Median progression-free survival and overall survival were 4.4 months (95% confidence interval [CI] = 2.7-5.2) and 8.1 months (95% CI = 5.1-12.0), respectively. CONCLUSIONS: A recommended and effective dose of vemurafenib for patients aged 12-17 years with metastatic or unresectable melanoma was not identified. Extremely low enrollment in this trial highlights the importance of considering the inclusion of adolescents with adult cancers in adult trials

Development and validation of a 30-day mortality index based on pre-existing medical administrative data from 13,323 COVID-19 patients: The Veterans Health Administration COVID-19 (VACO) Index.

BACKGROUND: Available COVID-19 mortality indices are limited to acute inpatient data. Using nationwide medical administrative data available prior to SARS-CoV-2 infection from the US Veterans Health Administration (VA), we developed the VA COVID-19 (VACO) 30-day mortality index and validated the index in two independent, prospective samples. METHODS AND FINDINGS: We reviewed SARS-CoV-2 testing results within the VA between February 8 and August 18, 2020. The sample was split into a development cohort (test positive between March 2 and April 15, 2020), an early validation cohort (test positive between April 16 and May 18, 2020), and a late validation cohort (test positive between May 19 and July 19, 2020). Our logistic regression model in the development cohort considered demographics (age, sex, race/ethnicity), and pre-existing medical conditions and the Charlson Comorbidity Index (CCI) derived from ICD-10 diagnosis codes. Weights were fixed to create the VACO Index that was then validated by comparing area under receiver operating characteristic curves (AUC) in the early and late validation cohorts and among important validation cohort subgroups defined by sex, race/ethnicity, and geographic region. We also evaluated calibration curves and the range of predictions generated within age categories. 13,323 individuals tested positive for SARS-CoV-2 (median age: 63 years; 91% male; 42% non-Hispanic Black). We observed 480/3,681 (13%) deaths in development, 253/2,151 (12%) deaths in the early validation cohort, and 403/7,491 (5%) deaths in the late validation cohort. Age, multimorbidity described with CCI, and a history of myocardial infarction or peripheral vascular disease were independently associated with mortality-no other individual comorbid diagnosis provided additional information. The VACO Index discriminated mortality in development (AUC = 0.79, 95% CI: 0.77-0.81), and in early (AUC = 0.81 95% CI: 0.78-0.83) and late (AUC = 0.84, 95% CI: 0.78-0.86) validation. The VACO Index allows personalized estimates of 30-day mortality after COVID-19 infection. For example, among those aged 60-64 years, overall mortality was estimated at 9% (95% CI: 6-11%). The Index further discriminated risk in this age stratum from 4% (95% CI: 3-7%) to 21% (95% CI: 12-31%), depending on sex and comorbid disease. CONCLUSION: Prior to infection, demographics and comorbid conditions can discriminate COVID-19 mortality risk overall and within age strata. The VACO Index reproducibly identified individuals at substantial risk of COVID-19 mortality who might consider continuing social distancing, despite relaxed state and local guidelines

Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor

Background : The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had. Methods : OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed. Results : OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4. Conclusions : Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications.This work was fnancially supported by the Ministry of Science and ICT (NRF2021R1A2C2008821). The Institute of Engineering Research at Seoul National University provided research facilities for this work

Identification and Functional Validation of the Novel Antimalarial Resistance Locus PF10_0355 in Plasmodium falciparum

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (~1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.Bill & Melinda Gates FoundationEllison Medical FoundationExxon Mobil FoundationFogarty International CenterNational Institute of Allergy and Infectious Diseases (U.S.)Burroughs Wellcome FundDavid & Lucile Packard FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra