Nuclear roles for non-lamin intermediate filament proteins.

The nuclear-localized lamins have long been thought to be the only intermediate filaments (IFs) with an impact on the architecture, properties, and functions of the nucleus. Recent studies, however, uncovered significant roles for IFs other than lamins (here referred to as "non-lamin IFs") in regulating key properties of the nucleus in various cell types and biological settings. In the cytoplasm, IFs often occur in the perinuclear space where they contribute to local stiffness and impact the shape and/or the integrity of the nucleus, particularly in cells under stress. In addition, selective non-lamin IF proteins can occur inside the nucleus where they partake in fundamental processes including nuclear architecture and chromatin organization, regulation of gene expression, cell cycle progression, and the repair of DNA damage. This text reviews the evidence supporting a role for non-lamin IF proteins in regulating various properties of the nucleus and highlights opportunities for further study.

Determinants of complementary feeding practices among children aged 6-24 months in urban slums of Pune, Maharashtra, in India.

BACKGROUND: Inequalities in child feeding practices are evident in urban slums in developing nations. Our study identified the determinants of complementary feeding (CF) practices in the informal settings of Pune, India, a district close to the business capital of India. METHODS: Employing a cross-sectional study design, 1066 mother-children dyads were surveyed. Five indicators defined by the WHO were used to study complementary feeding practices. Determinants of complementary feeding practices were identified using multivariate analyses. RESULTS: Timely initiation of CF was reported by 42%. Minimum acceptable diet (MAD), minimum meal frequency (MMF), and Diet Diversity Score > 4 were achieved by 14.9%, 76.5%, and 16.4%, respectively. Continued breastfeeding (CBF) at 2 years, and feeding processed foods were practiced by 94% and 50%, respectively. Among the maternal characteristics, a mother's age > 30 years at pregnancy was less likely to achieve DD [AOR: 0.195 (CI 0.047-0.809)] and MAD [AOR: 0.231 (CI 0.056-0.960)]. Mothers with lower education were less likely to meet MMF [AOR: 0.302 (0.113-0.807)], MAD [AOR: 0.505 (CI 0.295-0.867)] and to introduce formula feeds (FF) [AOR: 0.417 (0.193- 0.899)]. Among obstetric characteristics, birth spacing < 33 months was less likely to achieve DD [AOR: 0.594 (CI 0.365-0.965)] and CBF [AOR: 0.562 (CI: 0.322-0.982)]. Receiving IYCF counseling only during postnatal care hindered the timely initiation of CF [AOR: 0.638 (0.415-0.981)]. Very Low Birth Weight increased the odds of achieving DD [AOR: 2.384 (1.007-5.644)] and MAD [AOR: 2.588(CI: 1.054-6.352)], while low birth weight increased the odds of children being introduced to processed foods [AOR: 1.370 (CI: 1.056-1.776)]. Concerning socio-economic status, being above the poverty line increased the odds of achieving MMF, [AOR: 1.851 (1.005-3.407)]. Other backward castes showed higher odds of achieving MAD [AOR: 2.191 (1.208-3.973)] and undisclosed caste in our study setting decreased the odds of FF [AOR: 0.339 (0.170-0.677)]. Bottle feeding interfered with MMF [AOR: 0.440 (0.317-0.611)] and CBF [AOR: 0.153 (0.105-0.224)]. CONCLUSION: Investing in maternal education and IYCF counseling during both ANC and PNC to provide nutritious complementary foods alongside addressing poverty should be a national priority to prevent the double burden of undernutrition at an early age in informal settings.

Is Infant and Young Child-feeding (IYCF) a potential double-duty strategy to prevent the double burden of malnutrition among children at the critical age? Evidence of association from urban slums in Pune, Maharashtra, India.

BACKGROUND: This study characterized undernutrition among children (0-24 months) by age groups specified for Infant and Young Child-feeding (IYCF) and determined the association between child malnutrition and IYCF. METHODS: This cross-sectional survey recruited mother-children dyads (N = 1443). WHO standards were used to assess nutritional status and IYCF indicators. Multivariate analyses were performed to assess the association between IYCF and nutritional indicators. RESULTS: Stunting, underweight, wasting, overweight, and obesity were prevalent in 33.1%, 26%, 20.2%, 4.6%, and 2.9% of the children, respectively. Age-wise distribution of undernutrition identified severity of stunting and underweight at 10-24 months (median < -1.6 SD; < -1.2 SD; 25th percentile at -2.6 & -2.2 SD respectively) and wasting highest at 0-6 months (25th percentile close to -2SD). Boys manifested higher stunting (lower value -5.2 SD) and were more wasted (lower value -4.7 SD). IYCF prevalence recorded early initiation at 45.2%, exclusive breastfeeding at 23.1%, and prelacteal and bottle-feeding at 37.5 and 22.5% respectively. Child minimum diet diversity (MDD) ≥4 was not achieved by 84%. Minimum meal frequency and minimum acceptable diet were achieved by 75% and 14% respectively. Bottle-feeding increased the odds of wasting [AOR: 1.501 (95% CI: 1.062-2.121)], severe stunting [AOR: 1.595 (95% CI: 1.079-2.358)] and underweight [AOR: 1.519 (95% CI 1.102-2.094)]. Wasting according to BAZ scores was associated with delayed initiation of breastfeeding [AOR: 1.387 (95% CI: 1.018-1.889)] and bottle feeding [AOR: 1.538 (95% CI: 1.087-2.175)]. Delayed introduction of complementary feeding increased the odds of severe stunting [AOR: 2.189 (95% CI: 1.090-4.399)]. Formula feeding increased the odds of underweight [AOR: 1.738 (95% CI: 1.046-2.888)] and obesity [AOR: 4.664 (95% CI: 1.351-16.10)]. Prelacteal feeding increased the odds of severe forms of stunting and underweight by 56% and 79% respectively, and overweight by 96%. CONCLUSION: Setting and age-specific interventions to improve age-appropriate child-feeding practices are vital to address the double burden of malnutrition in the critical age group.

Revisiting the significance of keratin expression in complex epithelia.

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells and biological context (e.g. normal versus diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single-cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Furthermore, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic versus a post-mitotic differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia.

Correction to "Direct Visualization of Crystalline Domains in Carboxylated Nanocellulose Fibers".

[This corrects the article DOI: 10.1021/acsomega.0c00410.].

Sulphur-doped graphene quantum dot based fluorescent turn-on aptasensor for selective and ultrasensitive detection of omethoate.

Development of selective, ultra-sensitive, rapid and facile methods for the detection of chemical residues of toxic pesticides and hazardous chemicals are quite important in food safety, environmental monitoring and safeguarding public health. Herein, we presented a fluorescent turn-on aptasensor based on sulphur-doped graphene quantum dot (S-GQD) utilizing specific recognition and binding property of aptamer for the ultra-sensitive and selective detection of omethoate (OM) which is a systemic organophosphorus pesticide. The detection method is based on tuning aggregation-disaggregation mechanism of S-GQD by way of conformational alteration of the recognition probe. Fluorescence 'turn-on' process includes aggregation-induced quenching of S-GQD with aptamer via S-GQD-aptamer complex formation and its subsequent fluorescence recovery with the addition of OM by structural switching of S-GQD-aptamer complex to aptamer-omethoate complex. The reported 'switch-on' aptasensor has exhibited a low limit of detection of 0.001 ppm with high selectivity for OM over other pesticides.

A role for keratin 17 during DNA damage response and tumor initiation.

High levels of the intermediate filament protein keratin 17 (K17) are associated with poor prognoses for several human carcinomas. Studies in mouse models have shown that K17 expression is positively associated with growth, survival, and inflammation in skin and that lack of K17 delays onset of tumorigenesis. K17 occurs in the nucleus of human and mouse tumor keratinocytes where it impacts chromatin architecture, gene expression, and cell proliferation. We report here that K17 is induced following DNA damage and promotes keratinocyte survival. The presence of nuclear K17 is required at an early stage of the double-stranded break (DSB) arm of the DNA damage and repair (DDR) cascade, consistent with its ability to associate with key DDR effectors, including γ-H2A.X, 53BP1, and DNA-PKcs. Mice lacking K17 or with attenuated K17 nuclear import showed curtailed initiation in a two-step skin carcinogenesis paradigm. The impact of nuclear-localized K17 on DDR and cell survival provides a basis for the link between K17 induction and poor clinical outcomes for several human carcinomas.

Prevalence and Determinants of Early Initiation (EI), Exclusive Breastfeeding (EBF), and Prelacteal Feeding among Children Aged 0-24 Months in Slums of Pune City, in Maharashtra.

Infant and young child feeding practices remain a public health challenge in India. We determined the socio-demographic risk factors for early initiation, exclusive breastfeeding and prelacteal feeding in the urban slums of Pune city.A cross sectional survey of mother (N=1443) children (< 2 years) dyads was performed. Socio-demographic, maternal and child characteristics were recorded. Breastfeeding practices were assessed using WHO indicators. Multiple logistic regression was employed to model associations between socio-demographic factors and breastfeeding indicators.Early initiation was reported by 45.2%, prelacteal feeding by 37.5% and exclusive breastfeeding by 23.7%. Caesarean delivery decreased the odds of early initiation (AOR: 0.403; 95% CI; 0.303.-0.536) and exclusive breastfeeding (OR: 0.675; 95% CI: 0. 478-0.953), while it increased the odds of prelacteal feeding (AOR: 3.525; 95% CI: 2.653-4.683). Delivery in a public health care facility increased the odds of early initiation (AOR: 1.439; 95% CI: 1.095-1.891) and exclusive breastfeeding (OR: 0.514; 95% CI: 0.366-0.720), while it decreased the odds of prelacteal feeding (AOR: 0.421; 95% CI: 0.318-0.559). Odds of early initiation decreased significantly in very low-birth-weight (AOR: 0.209; CI: 0.76-0.567) whereas, it increased odds of prelacteal feeding (AOR: 1.389; 95% CI: 0.640-3.019), (AOR: 0.483; 95% CI: 0.262-0.889). Religion other than Hindu or Muslim, age of the mother between 26-30 years increased the odds of exclusive breastfeeding and parity <2 increased the odds of prelacteal feeding.Interventions that address setting specific determinants, focusing on local contexts are essential to improve child feeding practices in urban slums.

Keratin 17 regulates nuclear morphology and chromatin organization.

Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17-null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2ß (an isoform encoded by TMPO) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells.This article has an associated First Person interview with the first author of the paper.

Direct Visualization of Crystalline Domains in Carboxylated Nanocellulose Fibers.

Direct visualization of soft organic molecules like cellulose is extremely challenging under a high-energy electron beam. Herein, we adopt two ionization damage extenuation strategies to visualize the lattice arrangements of the ß-(1→4)-d-glucan chains in carboxylated nanocellulose fibers (C-NCFs) having cellulose II crystalline phase using high-resolution transmission electron microscopy. Direct imaging of individual nanocellulose fibrils with high-resolution and least damage under high-energy electron beam is achieved by employing reduced graphene oxide, a conducting material with high electron transmittance and Ag+ ions, with high electron density, eliminating the use of sample-specific, toxic staining agents, or other advanced add-on techniques. Furthermore, the imaging of cellulose lattices in a C-NCF/TiO2 nanohybrid system is accomplished in the presence of Ag+ ions in a medium revealing the mode of association of C-NCFs in the system, which validates the feasibility of the presented strategy. The methods adopted here can provide further understanding of the fine structures of carboxylated nanocellulose fibrils for studying their structure-property relationship for various applications.

Niche-Specific Factors Dynamically Regulate Sebaceous Gland Stem Cells in the Skin.

Oil-secreting sebaceous glands (SGs) are critical for proper skin function; however, it remains unclear how different factors act together to modulate SG stem cells. Here, we provide functional evidence that each SG lobe is serviced by its own dedicated stem cell population. Upon ablating Notch signaling in different skin subcompartments, we find that this pathway exerts dual counteracting effects on SGs. Suppressing Notch in SG progenitors traps them in a hybrid state where stem and differentiation features become intermingled. In contrast, ablating Notch outside of the SG stem cell compartment indirectly drives SG expansion. Finally, we report that a K14:K5→K14:K79 keratin shift occurs during SG differentiation. Deleting K79 destabilizes K14 in sebocytes, and attenuates SGs and eyelid meibomian glands, leading to corneal ulceration. Altogether, our findings demonstrate that SGs integrate diverse signals from different niches and suggest that mutations incurred within one stem cell compartment can indirectly influence another.

Clampdown of inflammation in aging and anticancer therapies by limiting upregulation and activation of GPCR, CXCR4.

One of the major pathological outcomes of DNA damage during aging or anticancer therapy is enhanced inflammation. However, the underlying signaling mechanism that drives this is not well understood. Here, we show that in response to DNA damage, ubiquitously expressed GPCR, CXCR4 is upregulated through the ATM kinase-HIF1α dependent DNA damage response (DDR) signaling, and enhances inflammatory response when activated by its ligand, chemokine CXCL12. A pharmacologically active compound screen revealed that this increased inflammation is dependent on reduction in cAMP levels achieved through activation of Gαi through CXCR4 receptor and PDE4A. Through in vivo analysis in mice where DNA damage was induced by irradiation, we validated that CXCR4 is induced systemically after DNA damage and inhibition of its activity or its induction blocked inflammation as well as tissue injury. We thus report a unique DNA damage-linked inflammatory cascade, which is mediated by expression level changes in a GPCR and can be targeted to counteract inflammation during anticancer therapies as well as aging.

ATM-ROS-iNOS axis regulates nitric oxide mediated cellular senescence.

Cellular senescence is an outcome of the accumulation of DNA damage which induces the growth arrest in cells. Physiologically, it is presumed to be mediated by accumulation of reactive oxygen species (ROS). Here, we show that another free radical, nitric oxide (NO) produced during inflammation or present as an environmental pollutant can also induce cellular senescence. In primary cells and various immortalized cell lines, exposure to chronic NO, through external addition or internally generated by iNOS expression, leads to the activation of DNA damage response and causes cellular senescence. The phenotype generated by NO includes robust growth arrest, increase in the levels of the DNA damage foci, ROS, SAß-gal staining, and inflammatory cytokines like IL-6 and IL-8, all hallmarks of cellular senescence similar to replicative senescence. Mechanistically, inhibitor and knockdown analysis revealed that NO mediates senescence through ATM kinase activation and the viability of cells is dependent on both ROS and ATM kinase involving the ATM-ROS-iNOS axis. Overall, we demonstrate that nitric oxide mediates cellular senescence through a novel free radical dependent genotoxic stress pathway.

Rapid, Acid-Free Synthesis of High-Quality Graphene Quantum Dots for Aggregation Induced Sensing of Metal Ions and Bioimaging.

Graphene quantum dots (GQDs) are zero-dimensional materials that exhibit characteristics of both graphene and quantum dots. Herein, we report a rapid, relatively green, one-pot synthesis of size-tunable GQDs from graphene oxide (GO) by a sonochemical method with intermittent microwave heating, keeping the reaction temperature constant at 90 °C. The GQDs were synthesized by oxidative cutting of GO using KMnO4 as an oxidizing agent within a short span of time (30 min) in an acid-free condition. The synthesized GQDs were of high quality and exhibited good quantum yield (23.8%), high product yield (>75%), and lower cytotoxicity (tested up to 1000 µg/mL). Furthermore, the as-synthesized GQDs were demonstrated as excellent fluorescent probes for bioimaging and label-free sensing of Fe(III) ions, with a detection limit as low as 10 × 10-6 M.

A molecular beacon-based DNA switch for reversible pH sensing in vesicles and live cells.

In this Communication, a molecular beacon-based DNA switch (LMB) is developed as an efficient and reversible pH sensing probe. Remarkably, LMB exhibited reversible structural transition between the closed (molecular beacon) and open (A-motif) states very efficiently in synthetic vesicles and live cells without the need for any transfection agents.

Stimuli-responsive colorimetric and NIR fluorescence combination probe for selective reporting of cellular hydrogen peroxide.

Hydrogen peroxide (H2O2) is a key reactive oxygen species and a messenger in cellular signal transduction apart from playing a vital role in many biological processes in living organisms. In this article, we present phenyl boronic acid-functionalized quinone-cyanine (QCy-BA) in combination with AT-rich DNA (exogenous or endogenous cellular DNA), i.e., QCy-BA⊂DNA as a stimuli-responsive NIR fluorescence probe for measuring in vitro levels of H2O2. In response to cellular H2O2 stimulus, QCy-BA converts into QCy-DT, a one-donor-two-acceptor (D2A) system that exhibits switch-on NIR fluorescence upon binding to the DNA minor groove. Fluorescence studies on the combination probe QCy-BA⊂DNA showed strong NIR fluorescence selectively in the presence of H2O2. Furthermore, glucose oxidase (GOx) assay confirmed the high efficiency of the combination probe QCy-BA⊂DNA for probing H2O2 generated in situ through GOx-mediated glucose oxidation. Quantitative analysis through fluorescence plate reader, flow cytometry and live imaging approaches showed that QCy-BA is a promising probe to detect the normal as well as elevated levels of H2O2 produced by EGF/Nox pathways and post-genotoxic stress in both primary and senescent cells. Overall, QCy-BA, in combination with exogenous or cellular DNA, is a versatile probe to quantify and image H2O2 in normal and disease-associated cells.

Temporally distinct roles of ATM and ROS in genotoxic-stress-dependent induction and maintenance of cellular senescence.

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM-ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

Chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counter-regulation.

Intratumoral delivery of IL-12 and GM-CSF induces local and systemic antitumor CD8(+) T cell activation and tumor kill. However, the effector response is transient and is rapidly countered by CD4(+) Foxp3(+) T suppressor cell expansion. To determine whether depletion of the pre-existing T suppressor cell pool prior to treatment could diminish posttherapy regulatory cell resurgence, FVBneuN mice bearing advanced spontaneous mammary tumors were treated with cyclophosphamide (CY) 1 d before IL-12/GM-CSF therapy. Administration of CY mediated a significant delay in the post-IL-12/GM-CSF T suppressor cell rebound, resulting in a 7-fold increase in the CD8(+) CTL/T suppressor cell ratio, a 3-fold enhancement of CTL cytotoxicity, and an extension of the effector window from 3 to 7 d. In long-term therapy studies, chronic chemoimmunotherapy promoted a dramatic enhancement of tumor regression, resulting in complete cure in 44% of the mice receiving CY plus IL-12/GM-CSF. Tumor eradication in the chronic therapy setting was associated with the ability to repeatedly rescue and maintain cytotoxic CD8(+) T cell activity. These findings demonstrated that chronic administration of CY in conjunction with immune therapy enhances the initial induction of antitumor T effector cells and, more importantly, sustains their cytotoxic activity over the long-term via persistent blockade of homeostatic counter-regulation.

Discovering the elusive underlying cause of a bilateral effusion combined with ascites.

A 77-year-old Asian man presented to the emergency department with bilateral pleural effusion and ascites accompanied with generalized weakness, dyspnea, tachycardia, and tachypnea. After an extensive workup that ruled out heart failure, pulmonary embolism, pneumonia, and malignancy-including extensive laboratory tests, electrocardiograms, chest x-ray, computed tomographic angiogram, computed tomography scans of the abdomen and pelvis, colonoscopy, thoracentesis, paracentesis, and exploratory laparoscopy-an elusive peritoneal tuberculosis was successfully identified. This case suggests that clinicians should consider extrapulmonary tuberculosis in their practice, given increasing immigration and the variety of populations present in our society. When tuberculosis is suspected, a negative smear for acid-fast bacillus, a lack of granulomas on histopathology, and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis. Exploratory laparoscopy or minilaparotomy has a high level of sensitivity and specificity so should be considered.