Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Simultaneous targeting of peripheral and brain tumors with a therapeutic nanoparticle to disrupt metabolic adaptability at both sites.

Brain metastasis of advanced breast cancer often results in deleterious consequences. Metastases to the brain lead to significant challenges in treatment options, as the blood-brain barrier (BBB) prevents conventional therapy. Thus, we hypothesized that creation of a nanoparticle (NP) that distributes to both primary tumor site and across the BBB for secondary brain tumor can be extremely beneficial. Here, we report a simple targeting strategy to attack both the primary breast and secondary brain tumors utilizing a single NP platform. The nature of these mitochondrion-targeted, BBB-penetrating NPs allow for simultaneous targeting and drug delivery to the hyperpolarized mitochondrial membrane of the extracranial primary tumor site in addition to tumors at the brain. By utilizing a combination of such dual anatomical distributing NPs loaded with therapeutics, we demonstrate a proof-of-concept idea to combat the increased metabolic plasticity of brain metastases by lowering two major energy sources, oxidative phosphorylation (OXPHOS) and glycolysis. By utilizing complementary studies and genomic analyses, we demonstrate the utility of a chemotherapeutic prodrug to decrease OXPHOS and glycolysis by pairing with a NP loaded with pyruvate dehydrogenase kinase 1 inhibitor. Decreasing glycolysis aims to combat the metabolic flexibility of both primary and secondary tumors for therapeutic outcome. We also address the in vivo safety parameters by addressing peripheral neuropathy and neurobehavior outcomes. Our results also demonstrate that this combination therapeutic approach utilizes mitochondrial genome targeting strategy to overcome DNA repair-based chemoresistance mechanisms.

BEND3 safeguards pluripotency by repressing differentiation-associated genes.

BEN domain-containing proteins are emerging rapidly as an important class of factors involved in modulating gene expression, yet the molecular basis of how they regulate chromatin function and transcription remains to be established. BEND3 is a quadruple BEN domain-containing protein that associates with heterochromatin and functions as a transcriptional repressor. We find that BEND3 is highly expressed in pluripotent cells, and the induction of differentiation results in the down-regulation of BEND3. The removal of BEND3 from pluripotent cells results in cells exhibiting upregulation of the differentiation-inducing gene expression signature. We find that BEND3 binds to the promoters of differentiation-associated factors and key cell cycle regulators, including CDKN1A, encoding the cell cycle inhibitor p21, and represses the expression of differentiation-associated genes by enhancing H3K27me3 decoration at these promoters. Our results support a model in which transcription repression mediated by BEND3 is essential for normal development and to prevent differentiation.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Hidradenitis suppurativa: Current understanding, diagnostic and surgical challenges, and developments in ultrasound application.

BACKGROUND: Hidradenitis suppurativa (HS) is debilitating, costly, chronic disease for which no cure exists and which often precipitates greater health concerns. While we are making advances in understanding, HS remains an area of attention which is evidenced by a 400% increase in research studies involving HS in the past 5 years. This includes research regarding the advantages and limitations of ultrasound (US) imaging and its ability to enhance the surgical treatment and medical management of HS. Herein, we describe the diagnostic and surgical obstacles that HS presents, the foremost of which is detection of subclinical information, and perform an in-depth synthesis of current knowledge regarding the use of US imaging to mitigate these obstacles. MATERIALS AND METHODS: A comprehensive literature review of US imaging in HS patients and a supplementary review of the current state of HS were conducted. CONCLUSION: Ultrasound imaging is a powerful tool in the diagnosis, monitoring, clinical management, and preoperative assessment of HS. However, it also has relevant limitations that necessitate additional consideration. SIGNIFICANCE: Hidradenitis suppurativa is a disabling skin disease that presents a diagnostic and surgical challenge. The invaluable advantages and relevant limitations that US imaging offers are beginning to be understood, leading to standardization and increased implementation. US imaging has the potential to drastically improve patient care and merits further attention.

A cross-sectional study: medication safety among cancer in-patients in tertiary care hospitals in KPK, Pakistan.

BACKGROUND: Medication safety in cancer patients receiving complex medication regimens is an important problem in various settings. Medication related events, interceptions and interventions are not well described in this area. We intended to study incidence, types, settings and stages involved, root cause analysis, medication classes involved and the level of harm cause by medication errors in two hospitals providing oncology services comparatively. The severity of incidents and interventions are studied. METHODS: It was a prospective cross sectional study among cancer in-patients of two tertiary care hospitals of KPK. Scale by NCC-MERP was used for evaluation of all medication related incidents. The data obtained was analyzed by IBM SPSS statistics 22 with 95% confidence interval and used the same for other descriptive statistics. RESULTS: All medication orders were reviewed at both sites (Computerized Prescription Order Entry and HWP systems). Potential ADEs incidence was found high at site 2 (97.5%) while medication errors without harm was high at site 1 (97.5%). Most events occur at prescribing level 87.6 and 81.7% at both sites 1 and 2. Types highly reported involved improper dose 31.4 and 15.5%, monitoring error 14.6 and 15.2% at site 1 and 2. Medications involved in these incidents were antibiotics 44 and 12.7%, antiemetic 7.5 and 15.8% and antineoplastic 2.9 and 9.4% at site 1 and 2. Severity of 3.6 and 36.5% incidents had potential to cause harm at site 1 and 2. Root causes were human factors 62.6 and 72.3%, drug selection 33.6 and 38.8%, and dose selection 39.6 and 15.3% at sites 1 and 2. Contributing factors including staff training 33.6 and 24.3%, system for covering patient care 14.9 and 36.6%, communication system 2.4 and 20.3%, interruptions 9.7 and 7.3% and others 78.8 and 68.6% were highly reported. Preventability of medication errors was 99% at both sites. Intervention was taken in 90.5% events at site 1 (CPOE system) while the incidence lowest at site 2 (HWP system). CONCLUSION: Medication related events are high among cancer in-patients at the site lacking updated electronic system for medication prescribing. Proper training about medication safety, reporting and interventions are required.

Photoacoustic/Ultrasound/Optical Coherence Tomography Evaluation of Melanoma Lesion and Healthy Skin in a Swine Model.

The marked increase in the incidence of melanoma coupled with the rapid drop in the survival rate after metastasis has promoted the investigation into improved diagnostic methods for melanoma. High-frequency ultrasound (US), optical coherence tomography (OCT), and photoacoustic imaging (PAI) are three potential modalities that can assist a dermatologist by providing extra information beyond dermoscopic features. In this study, we imaged a swine model with spontaneous melanoma using these modalities and compared the images with images of nearby healthy skin. Histology images were used for validation.

Radioprotective Agents: Strategies and Translational Advances.

Radioprotectors are agents required to protect biological system exposed to radiation, either naturally or through radiation leakage, and they protect normal cells from radiation injury in cancer patients undergoing radiotherapy. It is imperative to study radioprotectors and their mechanism of action comprehensively, looking at their potential therapeutic applications. This review intimately chronicles the rich intellectual, pharmacological story of natural and synthetic radioprotectors. A continuous effort is going on by researchers to develop clinically promising radioprotective agents. In this article, for the first time we have discussed the impact of radioprotectors on different signaling pathways in cells, which will create a basis for scientific community working in this area to develop novel molecules with better therapeutic efficacy. The bright future of exceptionally noncytotoxic derivatives of bisbenzimidazoles is also described as radiomodulators. Amifostine, an effective radioprotectant, has been approved by the FDA for limited clinical use. However, due to its adverse side effects, it is not routinely used clinically. Recently, CBLB502 and several analog of a peptide are under clinical trial and showed high success against radiotherapy in cancer. This article reviews the different types of radioprotective agents with emphasis on the strategies for the development of novel radioprotectors for drug development. In addition, direction for future strategies relevant to the development of radioprotectors is also addressed.

Early Onset Diabetes - Genetic And Hormonal Analysis In Pakistani Population.

BACKGROUND: Mitochondrial DNA mutation and hormonal imbalance is involved in the pathogenesis of early onset diabetes but data is lacking in Pakistani population. The study was planned to delineate the clinical presentation of early onset diabetes with possible hormonal and genetic etiological factors and aascertain the possible etiological role of insulin and glucagon in these patients either on oral hypoglycaemic or subcutaneous insulin therapy. METHODS: Retrospective, analytical case control study with conventional sampling technique carried at Centre for Research in Experimental and Applied Medicine (CREAM) affiliated with the department of Biochemistry and Molecular Biology, Army Medical College Rawalpindi from Dec 2006 to July 2011. Study included the patients (20-35 years of age) with early onset diabetes on oral hypoglycemic (n=240), insulin therapy (n=280), and compared with non-diabetic healthy controls (n=150). A fragment surrounding tRNALeu (UUR) gene was amplified by AmpliTaq from mtDNA which was extracted from peripheral blood leucocytes. Then it was subjected to restriction endonucleases, ApaI for A3242G mutation and HaeIII for G3316A mutation detection. Plasma glucose, glycosylated Hb, osmolality, insulin and glucagon levels along with ABGs analysis was also done. RESULTS: Non diabetic controls comprised of 51% males and 49% females, diabetics on oral hypoglycemic 60% males and 40 % females and on insulin therapy 54% males and 46% females. Insulin dependent diabetics had statistically significant hyperglucagonemia, acidemia and bicarbonate deficit. MtDNA A3242G and G3316A mutations were not detected. CONCLUSIONS: relative hyperglucagonemia and acidemia in Insulin dependent diabetics was a potent threat leading to DKA. The absence of two mtDNA mutations in ND1 gene rules out the possibility of involvement of these mutations in early onset diabetes in Pakistani population.

Intracellular locations of replication proteins and the origin of replication during chromosome duplication in the slowly growing human pathogen Helicobacter pylori.

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.

Role of Transcription Factor BEND3 and Its Potential Effect on Cancer Progression.

BEND3 is a transcription factor that plays a critical role in the regulation of gene expression in mammals. While there is limited research on the role of BEND3 as a tumor suppressor or an oncogene and its potential role in cancer therapy is still emerging, several studies suggest that it may be involved in both the processes. Its interaction and regulation with multiple other factors via p21 have already been reported to play a significant role in cancer development, which serves as an indication of its potential role in oncogenesis. Its interaction with chromatin modifiers such as NuRD and NoRC and its role in the recruitment of polycomb repressive complex 2 (PRC2) are some of the additional events indicative of its potential role in cancer development. Moreover, a few recent studies indicate BEND3 as a potential target for cancer therapy. Since the specific mechanisms by which BEND3 may contribute to cancer progression are not yet fully elucidated, in this review, we have discussed the possible pathways BEND3 may take to serve as an oncogenic driver or suppressor.

Cell-of-Origin Targeted Drug Repurposing for Triple-Negative and Inflammatory Breast Carcinoma with HDAC and HSP90 Inhibitors Combined with Niclosamide.

We recently identified a cell-of-origin-specific mRNA signature associated with metastasis and poor outcome in triple-negative carcinoma (TNBC). This TNBC cell-of-origin signature is associated with the over-expression of histone deacetylases and zinc finger protein HDAC1, HDAC7, and ZNF92, respectively. Based on this signature, we discovered that the combination of three drugs (an HDAC inhibitor, an anti-helminthic Niclosamide, and an antibiotic Tanespimycin that inhibits HSP90) synergistically reduces the proliferation of the twelve tested TNBC cell lines. Additionally, we discovered that four out of five inflammatory breast carcinoma cell lines are sensitive to this combination. Significantly, the concentration of the drugs that are used in these experiments are within or below clinically achievable dose, and the synergistic activity only emerged when all three drugs were combined. Our results suggest that HDAC and HSP90 inhibitors combined with the tapeworm drug Niclosamide can achieve remarkably synergistic inhibition of TNBC and IBC. Since Niclosamide, HDAC, and HSP90 inhibitors were approved for clinical use for other cancer types, it may be possible to repurpose their combination for TNBC and IBC.

Determination of growth pattern of 7-12 years old children in YAZD city and comparison of it with WHO standards.

OBJECTIVE: To assess the growth pattern of children in Yazd, and compare it with standards outlined by the World Health Organisation. METHODS: The cross-sectional study, which was conducted in 2009, involved 20 guidance schools of Yazd city. A total of 1921 children were chosen by simple cluster method and general demographic and anthropometric information regarding the subjects was extracted from students' health identification booklets. Data was analysed through SPSS. RESULTS: The mean values of height in 7th and 12th years of age were 119 +/- 8.8cm and 150 +/- 6.6cm in boys and 117 +/- 5.97cm and 152 +/- 7.3cm in girls respectively. The mean values of weight in 7th and 12th years of age were 20 +/- 3.39kg and 39 +/- 1kg in boys and 20 +/- 3.78kg and 41 +/- 1.07kg in girls respectively. These values generally were below the values set by the World Health Organisation till the 9th year of age, and above them beyond the 9th year. CONCLUSION: The growth pattern among children in Yazd is better than some of the other provinces in Iran. Larger studies are needed for generating Iran-specific standard for better assessment of the growth and nutritional status of Iranian children.

ZNF92, an unexplored transcription factor with remarkably distinct breast cancer over-expression associated with prognosis and cell-of-origin.

Tumor phenotype is shaped both by transforming genomic alterations and the normal cell-of-origin. We identified a cell-of-origin associated prognostic gene expression signature, ET-9, that correlates with remarkably shorter overall and relapse free breast cancer survival, 8.7 and 6.2 years respectively. The genes associated with the ET-9 signature are regulated by histone deacetylase 7 (HDAC7) partly through ZNF92, a previously unexplored transcription factor with a single PubMed citation since its cloning in 1990s. Remarkably, ZNF92 is distinctively over-expressed in breast cancer compared to other tumor types, on a par with the breast cancer specificity of the estrogen receptor. Importantly, ET-9 signature appears to be independent of proliferation, and correlates with outcome in lymph-node positive, HER2+, post-chemotherapy and triple-negative breast cancers. These features distinguish ET-9 from existing breast cancer prognostic signatures that are generally related to proliferation and correlate with outcome in lymph-node negative, ER-positive, HER2-negative breast cancers. Our results suggest that ET-9 could be also utilized as a predictive signature to select patients for HDAC inhibitor treatment.

Glutamine metabolism and radiosensitivity: Beyond the Warburg effect.

Mounting data suggest that cancer cell metabolism can be utilized therapeutically to halt cell proliferation, metastasis and disease progression. Radiation therapy is a critical component of cancer treatment in curative and palliative settings. The use of metabolism-based therapeutics has become increasingly popular in combination with radiotherapy to overcome radioresistance. Over the past year, a focus on glutamine metabolism in the setting of cancer therapy has emerged. In this mini-review, we discuss several important ways (DNA damage repair, oxidative stress, epigenetic modification and immune modulation) glutamine metabolism drives cancer growth and progression, and present data that inhibition of glutamine utilization can lead to radiosensitization in preclinical models. Future research is needed in the clinical realm to determine whether glutamine antagonism is a feasible synergistic therapy that can be combined with radiotherapy.

GATA2 regulates mast cell identity and responsiveness to antigenic stimulation by promoting chromatin remodeling at super-enhancers.

Mast cells are critical effectors of allergic inflammation and protection against parasitic infections. We previously demonstrated that transcription factors GATA2 and MITF are the mast cell lineage-determining factors. However, it is unclear whether these lineage-determining factors regulate chromatin accessibility at mast cell enhancer regions. In this study, we demonstrate that GATA2 promotes chromatin accessibility at the super-enhancers of mast cell identity genes and primes both typical and super-enhancers at genes that respond to antigenic stimulation. We find that the number and densities of GATA2- but not MITF-bound sites at the super-enhancers are several folds higher than that at the typical enhancers. Our studies reveal that GATA2 promotes robust gene transcription to maintain mast cell identity and respond to antigenic stimulation by binding to super-enhancer regions with dense GATA2 binding sites available at key mast cell genes.

Brain-Accumulating Nanoparticles for Assisting Astrocytes to Reduce Human Immunodeficiency Virus and Drug Abuse-Induced Neuroinflammation and Oxidative Stress.

Human neurotropic immunodeficiency virus (HIV) ingress into the brain and its subsequent replication after infection results in viral reservoirs in the brain. The infected cells include microglia, perivascular macrophages, and astrocytes. HIV-associated neurocognitive disorders (HAND) affect glial cells by activating microglia and macrophages through neuroinflammation, as well as astrocytes through mitochondrial dysfunctions and the onset of oxidative stress, impairing the ability of these cells to engage in neuroprotection. Furthermore, the risk of neuroinflammation associated with HAND is magnified by recreational drug use in HIV-positive individuals. Most of the therapeutic options for HIV cannot be used to tackle the virus in the brain and treat HAND due to the inability of currently available combination antiretroviral therapies (ARTs) and neuroprotectants to cross the blood-brain barrier, even if the barrier is partially compromised by infection. Here, we report a strategy to deliver an optimized antiretroviral therapy combined with antioxidant and anti-inflammatory neuroprotectants using biodegradable brain-targeted polymeric nanoparticles to reduce the burden caused by viral reservoirs in the brain and tackle the oxidative stress and inflammation in astrocytes and microglia. Through in vitro coculture studies in human microglia and astrocytes as well as an in vivo efficacy study in an EcoHIV-infected, methamphetamine-exposed animal model, we established a nanoparticle-based therapeutic strategy with the ability to treat HIV infection in the central nervous system in conditions simulating drug use while providing enhanced protection to astrocytes, microglia, and neurons.

PL-7 Antisynthetase Syndrome in Association with Sjögren's, Systemic Lupus Erythematosus, and Rheumatoid Arthritis.

We present a rare case of PL-7 antisynthetase syndrome (ASS) in association with Sjögren's, systemic lupus erythematosus (SLE), and seropositive rheumatoid arthritis (RA). Initially, the patient was diagnosed with Sjögren's followed by Sjögren's/SLE overlap and then Sjögren's/SLE/RA overlap. She was eventually diagnosed with Sjögren's/SLE/RA overlap with PL-7 ASS with interstitial lung disease (ILD). ILD was discovered after complaints of pleuritic chest pain with subsequent workup with coronary computed tomography (CT) revealing pulmonary fibrosis. This case demonstrates the ambiguity with which symptoms of ASS can present; given the high respiratory morbidity and mortality of ASS especially in non-Jo-1 patients, those who present with Raynaud's, myositis, or joint pain, whether together or in isolation, should be assessed for presence of additional features of ASS and potentially undergo testing for ASS antibodies if appropriate.

Clinically Approved Antiviral Drug in an Orally Administrable Nanoparticle for COVID-19.

There is urgent therapeutic need for COVID-19, a disease for which there are currently no widely effective approved treatments and the emergency use authorized drugs do not result in significant and widespread patient improvement. The food and drug administration-approved drug ivermectin has long been shown to be both antihelmintic agent and a potent inhibitor of viruses such as Yellow Fever Virus. In this study, we highlight the potential of ivermectin packaged in an orally administrable nanoparticle that could serve as a vehicle to deliver a more potent therapeutic antiviral dose and demonstrate its efficacy to decrease expression of viral spike protein and its receptor angiotensin-converting enzyme 2 (ACE2), both of which are keys to lowering disease transmission rates. We also report that the targeted nanoparticle delivered ivermectin is able to inhibit the nuclear transport activities mediated through proteins such as importin α/ß1 heterodimer as a possible mechanism of action. This study sheds light on ivermectin-loaded, orally administrable, biodegradable nanoparticles to be a potential treatment option for the novel coronavirus through a multilevel inhibition. As both ACE2 targeting and the presence of spike protein are features shared among this class of virus, this platform technology has the potential to serve as a therapeutic tool not only for COVID-19 but for other coronavirus strains as well.

The S-phase-induced lncRNA SUNO1 promotes cell proliferation by controlling YAP1/Hippo signaling pathway.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.