Multispectral Imaging of Metabolic Fluorophores: Comparing In Vivo and Fresh Ex Vivo Tissue.

The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.

Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

Spesolimab: A Review of the First IL-36 Blocker Approved for Generalized Pustular Psoriasis.

OBJECTIVE: This article reviews clinical trial data that assesses the safety, efficacy, and clinical application of spesolimab, an interleukin-36 (IL-36) blocker, for the treatment of generalized pustular psoriasis (GPP). DATA SOURCES: A review of the literature was conducted using the search terms: "spesolimab," "BI 655130," and "spevigo" in MEDLINE (PubMed) and Clinicaltrials.gov from January 1, 1950 to October 31, 2023. STUDY SELECTION AND DATA EXTRACTION: Relevant articles in English relating to the pharmacodynamics, pharmacokinetics, efficacy, and safety of spesolimab were included. DATA SYNTHESIS: In one phase 2 clinical trial evaluating single dose IV spesolimab for GPP flares at day 8, 54% of patients receiving spesolimab had a GPP physician global assessment (GPPGA) pustulation subscore of 0, and 43% had a GPPGA total score of 0 compared with 6% and 11% for the placebo group, respectively. Another phase 2 clinical trial assessing subcutaneous spesolimab found 23% of patients in low-dose, 29% in medium-dose, and 10% of high-dose spesolimab had flares by week 48 compared with 52% of the placebo group. Hazard ratios for time to GPP flare compared with placebo were 0.16 (P = 0.0005), 0.35 (P = 0.0057), and 0.47 (P = 0.027) for the spesolimab groups, respectively. Infection rates were similar across treatment and placebo groups, and severe adverse events such as drug reactions with eosinophilia and systemic symptom (DRESS), cholelithiasis, and breast cancer occurred with spesolimab. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE IN COMPARISON TO EXISTING DRUGS: Spesolimab is a first-in-class IL-36 monoclonal antibody receptor antagonist approved for the treatment of acute GPP flares. It is a safe and effective therapeutic agent in preventing future GPP flares, with no current comparator trials with other GPP agents. CONCLUSION: Spesolimab is a safe and effective treatment for acute GPP flares in adults. Future clinical trials can establish safety and efficacy compared with other agents.

Hinfp is a guardian of the somatic genome by repressing transposable elements.

Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. We show here that loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of Drosophila after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of Hinfp in this process is Histone1, which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

Oral cancer patients achieve comparable survival at high safety-net burden hospitals.

PURPOSE: To evaluate the impact of hospital safety-net burden and social demographics on the overall survival of patients with oral cavity squamous cell carcinoma. MATERIALS AND METHODS: We identified 48,176 oral cancer patients diagnosed between the years 2004 to 2015 from the National Cancer Database and categorized treatment facilities as no, low, or high safety-net burden hospitals based on the percentage of uninsured or Medicaid patients treated. Using the Kaplan Meier method and multivariate analysis, we examined the effect of hospital safety-net burden, sociodemographic variables, and clinical factors on overall survival. RESULTS: Of the 1269 treatment facilities assessed, the median percentage of uninsured/Medicaid patients treated was 0% at no, 11.6% at low, and 23.5% at high safety-net burden hospitals and median survival was 68.6, 74.8, and 55.0 months, respectively (p < 0.0001). High safety-net burden hospitals treated more non-white populations (15.4%), lower median household income (<$30,000) (23.2%), and advanced stage cancers (AJCC III/IV) (54.6%). Patients treated at low (aHR = 0.97; 95% CI = 0.91-1.04, p = 0.405) and high (aHR = 1.05; 95% CI = 0.98-1.13, p = 0.175) safety-net burden hospitals did not experience worse survival outcomes compared to patients treated at no safety-net burden hospitals. CONCLUSION: High safety-net burden hospitals treated more oral cancer patients of lower socioeconomic status and advanced disease. Multivariate analysis showed high safety-net burden hospitals achieved comparable patient survival to lower burden hospitals.

Delayed onset of tooth decay in a routine pediatric adenotonsillectomy.

Adenotonsillectomy is a common pediatric surgical procedure with a well-defined safety profile. Major complications from this procedure include bleeding/hemorrhage, infection, pain leading to dehydration, and airway obstruction or edema. Though rare, oral endotracheal intubation and oral retractor placement may result in injuries to the teeth and the surrounding soft tissue. We describe a rare case of delayed tooth decay in a 3-year-old female following an otherwise routine adenotonsillectomy.

Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S-phase transition.

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3-dimensional context of nuclear organization is reflected by two well documented observations. DNA replication-dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone-related requirements for G1/S-phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci.

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression.

Maternal expression and early induction of histone gene transcription factor Hinfp sustains development in pre-implantation embryos.

Fidelity of histone gene expression is important for normal cell growth and differentiation that is stringently controlled during development but is compromised during tumorigenesis. Efficient production of histones for packaging newly replicated DNA is particularly important for proper cell division and epigenetic control during the initial pre-implantation stages of embryonic development. Here, we addressed the unresolved question of when the machinery for histone gene transcription is activated in the developing zygote to accommodate temporal demands for histone gene expression. We examined induction of Histone Nuclear Factor P (HINFP), the only known transcription factor required for histone H4 gene expression, that binds directly to a unique H4 promoter-specific element to regulate histone H4 transcription. We show that Hinfp gene transcripts are stored in oocytes and maternally transmitted to the zygote. Transcripts from the paternal Hinfp gene, which reflect induction of zygotic gene expression, are apparent at the 4- to 8-cell stage, when most maternal mRNA pools are depleted. Loss of Hinfp expression due to gene ablation reduces cell numbers in E3.5 stage embryos and compromises implantation. Reduced cell proliferation is attributable to severe reduction in histone mRNA levels accompanied by reduced cell survival and genomic damage as measured by cleaved Caspase 3 and phospho-H2AX staining, respectively. We conclude that transmission of maternal Hinfp transcripts and zygotic activation of the Hinfp gene together are necessary to control H4 gene expression in early pre-implantation embryos in order to support normal embryonic development.

Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle.

The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal, and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. J. Cell. Physiol. 232: 1254-1257, 2017. © 2016 Wiley Periodicals, Inc.

Precocious Phenotypic Transcription-Factor Expression During Early Development.

A novel role for phenotypic transcription factors in very early differentiation was recently observed and merits further study to elucidate what role this precocious expression may have in development. The RUNX1 transcription factor exhibits selective and transient upregulation during early mesenchymal differentiation. In contrast to phenotype-associated transcriptional control of gene expression to establish and sustain hematopoietic/myeloid lineage identity, precocious expression of RUNX1 is functionally linked to control of an epithelial to mesenchymal transition that is obligatory for development. This early RUNX1 expression spike provides a paradigm for precocious expression of a phenotypic transcription factor that invites detailed mechanistic study to fully understand its biological importance. J. Cell. Biochem. 118: 953-958, 2017. © 2016 Wiley Periodicals, Inc.

Lineage-Specific Early Differentiation of Human Embryonic Stem Cells Requires a G2 Cell Cycle Pause.

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775.

Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis.

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint is associated with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats, and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes, and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose, and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with P = e(-13) to e(-36) . Cell cycle expression networks show species, sex and tissue variability, and they are enriched in mRNA transcripts associated with mitosis, many of which are associated with chromosomal instability.

Spindle microtubule dysfunction and cancer predisposition.

Chromosome segregation and spindle microtubule dynamics are strictly coordinated during cell division in order to preserve genomic integrity. Alterations in the genome that affect microtubule stability and spindle assembly during mitosis may contribute to genomic instability and cancer predisposition, but directly testing this potential link poses a significant challenge. Germ-line mutations in tumor suppressor genes that predispose patients to cancer and alter spindle microtubule dynamics offer unique opportunities to investigate the relationship between spindle dysfunction and carcinogenesis. Mutations in two such tumor suppressors, adenomatous polyposis coli (APC) and Shwachman-Bodian-Diamond syndrome (SBDS), affect multifunctional proteins that have been well characterized for their roles in Wnt signaling and interphase ribosome assembly, respectively. Less understood, however, is how their shared involvement in stabilizing the microtubules that comprise the mitotic spindle contributes to cancer predisposition. Here, we briefly discuss the potential for mutations in APC and SBDS as informative tools for studying the impact of mitotic spindle dysfunction on cellular transformation.

Evaluation of probiotic milk on salivary mutans streptococci count: an in vivo microbiological study.

OBJECTIVE: The objective of the present study was to investigate the effect of probiotic Lactobacillus casei Shirota on the values of mutans streptococci counts in saliva. STUDY DESIGN: 31 children were included in this double blinded study. Study design included 4 periods which consisted of a run in period, two intervention periods and a washout period. During the intervention periods children were either given probiotic milk or control milk for 10 days. Pre and post intervention salivary samples were subjected to microbiological evaluation. Then numbers of mutans streptococci were taken by identifying colony morphology. RESULTS: A statistically significant reduction of mutans streptococci colony count was noted in the probiotic group (p=.003). The reduction in children with higher levels of mutans streptococci (105) after intervention was 34% in the probiotic group. CONCLUSION: Daily consumption of milk containing probiotic bacteria can reduce the levels of mutans streptococci and may contribute to the prevention of dental caries.

A Study of the Demographic Profile, Screening, and Management of Patients Visiting a Retinopathy of Prematurity Clinic.

Background and objective Retinopathy of prematurity (ROP) is a retinal vasoproliferative disease affecting premature infants. Despite improvements in neonatal care and management, ROP still remains a major cause of childhood blindness worldwide. Studying the demographic profile and screening is essential to develop predictive models, to gain insights into the cause of retinal vascular diseases and diseases of prematurity, and to determine the future management and research in ROP. The objective of the present study was to estimate the incidence of ROP, to identify the risk factors that predispose to ROP, and to assess the outcome of these cases. Hence, this study was conducted in a tertiary care hospital in Maharashtra. Method A prospective, observational study was conducted from 10 August 2022 to 10 October 2022. Infants with gestational ages < 34 weeks, birth weights < 2000 g, infants who received supplemental oxygen therapy, or patients who required NICU stay were screened for ROP. Demographic details were recorded to assess the risk factors and treatment was given according to the severity of ROP grade. Result A total of 160 eyes of 80 infants were screened and analysed. The overall incidence of "any ROP" was 19 patients (38 eyes), i.e., 24%. Out of 80 patients, six were of 28 weeks gestational age, of whom four (67%) were positive for ROP. The mean birth weight of infants with ROP was 1331.58 ± 238.532 g (p < 0.0001). ROP stage 1 was seen in five patients (26.32%), stage 2 in 10 patients (52.63%), and stage 3 in four patients (21.0%), with no subjects in stages 4 & 5. Out of 19 patients, six (32%) had type 1 ROP, and 13 (68%) had type 2 ROP. Out of 19 cases, 13 (68%) received follow-up care based on the severity of their disease, and six (32%) were treated with panretinal photocoagulation (PRP) laser. Conclusion Incidence of any ROP was 24%. Prematurity, low birth weight, and oxygen therapy remain the most significant risk factors associated with the development of ROP. Early referral, diagnosis, and timely intervention will play a monumental role in improving the prognosis of this potentially blinding disease.

LINC01638 Sustains Human Mesenchymal Stem Cell Self-Renewal and Competency for Osteogenic Cell Fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC cell growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2,000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.