Correction: Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages.

[This corrects the article DOI: 10.1371/journal.pgen.1009752.].

Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response.

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.

Artificial Modulation and Rewiring of Cell Cycle Progression Using Synthetic Circuits in Fission Yeast.

Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms that sustain cell proliferation and the ways in which they interact with other cellular pathways. In this context, the use of a synthetic approach to simplify the cell cycle network in unicellular genetic models such as fission yeast has opened the door to studying the biology of proliferating cells from unique perspectives. Here, we provide a series of methods based on a minimal cell cycle module in the fission yeast Schizosaccharomyces pombe that allows for an unprecedented artificial control of cell cycle events, enabling the rewiring and remodeling of cell cycle progression.

Fascial Arthroplastic Ossiculoplasty - A New Technique of Ossicular Reconstruction.

OBJECTIVE: Ossicular reconstruction with autologous materials has been practised for a long time, but the procedure may lead to stiffness of joints and bony ankyloses thus hampering the sound conduction mechanism. This paper describes a novel technique of creating joint capsule around the reconstructed joints with temporalis fascia strips hoping a firm and movable union with full functional restoration. MATERIALS & METHODS: This study is a prospective study of 35 patients requiring tympanomastoid surgery with ossicular reconstruction and were studied over a period of 2 years. The newly reconstructed ossicular joints were wrapped with temporalis fascia strips. Post-operatively air-bone gap closure were studied and statistically analysed. RESULTS: A significant air-bone gap closure was achieved in 23 patients, while moderate improvement was seen in remaining cases. Only 1 patient had tympanic membrane graft failure and rest all had well healed tympanic membrane. CONCLUSION: Fascial arthroplastic ossiculoplasty is a simple and safe procedure and the results are reproducible and more predictable. It provides an ossicular assembly which is near to normal in its physiological functions.

Long-term evolution of proliferating cells using the eVOLVER platform.

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new stand-alone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled set-up for long-term experimental evolution.

Long-term evolution of proliferating cells using the eVOLVER platform.

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently-developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new standalone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled setup for long-term experimental evolution.

Design and Characterization of Silver Nanoparticles of Different Species of Curcuma in the Treatment of Cancer Using Human Colon Cancer Cell Line (HT-29).

BACKGROUND: Cancer is a deadly disease responsible for worldwide mortality; usually, middle- and low-income countries have been more affected by cancer and are responsible for 70% of deaths. The present study was performed with the aim to design silver nanoparticles using three species of Curcuma, i.e., Curcuma longa, Curcuma aromatica, and Curcuma caesia. METHODS: The rhizomes of different plants were extracted with ethanol. The rhizome extracts were used to prepare silver nanoparticles. It was optimized at different pH, silver ion concentrations, and concentrations of plant extracts. The anticancer activity of prepared nanoparticles of C. longa, C. aromatica, and C. caesia was evaluated on a human colon cancer cell line (HT-29) using sulforhodamine B (SRB) assay. RESULTS: The percentage yield of C. longa, C. aromatica, and C. caesia was 11.34 g, 15.45 g, and 12.67 g, respectively. The results exhibited that the prepared nanoparticles were smooth and spherical. All the nanoparticles of rhizome extracts rescued the viability of HT-29 cells in a different extent. HT-29 cells were sensitive to prepared nanoparticles that induce more cytotoxicity towards cancer cells. CONCLUSION: Thus, the prepared silver nanoparticle of Curcuma species through green synthesis may help treat cancer with low toxicity.

Influence of periodontitis and diabetes on structure and cytokine content of platelet-rich fibrin.

BACKGROUND: Platelet-rich fibrin (PRF) is a second-generation platelet concentrate with multiple applications in wound healing and regeneration in both periodontitis and diabetes. However, the three dimensional (3-D) structure and cytokine content of PRF might be altered in patients suffering from either/both of the chronic inflammatory conditions, ultimately influencing the efficacy of PRF as a biomaterial for regenerative medicine. AIM: The aim of the present study was hence to evaluate the effect of both these chronic inflammatory diseases on the 3-D structure of PRF membrane. An attempt was also made to compare the growth factor content between the plasma and RBC ends of the prepared PRF gel. MATERIALS & METHODS: L-PRF was prepared for twenty participants, healthy (5), periodontitis (5), T2DM (5) and T2DM with periodontitis (5). Porosity and fiber diameter of PRF membranes was visualized under FE-SEM and measured using ImageJ Software. PDGF-BB and TGF-ß1 levels in PRF gel were assessed by ELISA. RESULTS: The average diameter of fibrin fibers under FE-SEM was 0.15 to 0.30 micrometers. Porosity was higher at the plasma end (p = 0.042). Red blood cell (RBC) end of the membrane had thinner fibers arranged in a comparatively more dense and compact structure with smaller porosities. Healthy subjects had the least porous PRF compared to subjects with either/both of the chronic conditions. PDGF-BB levels were similar along all the four groups. TGF-ß1 levels were highest in healthy subjects. DISCUSSION: 3-D structure and growth factor content of PRF are influenced by a person's periodontal and/or diabetic status. The RBC end of the PRF membrane, as compared to the plasma end, has thinner fibers arranged in a comparatively more dense and compact structure with smaller porosities, and hence should be favored during periodontal regenerative procedures. CONCLUSION: Both periodontitis and diabetes have a significant influence on the 3-D structure and growth factor content of PRF produced.

Inferring and perturbing cell fate regulomes in human brain organoids.

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.

Preparation of radiolabeled erlotinib analogues and analysis of the effect of linkers.

Erlotinib is a first generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) which was granted Food and Drug administration (FDA) approval for treatment of patients with locally advanced or metastatic NSCLC. The present study aimed at development of radiolabeled erlotinib variants as tyrosine kinase inhibitors. Three DOTA-erlotinib conjugates were prepared for radiolabeling with 177Lu. The terminal alkyne group of erlotinib was modified by performing Cu-catalyzed click chemistry and three different linkers were introduced which were then conjugated to the chelator, DOTA. The DOTA-erlotinib conjugates were characterized by 1H NMR and ESI-MS. 177Lu-DOTA-erlotinib complexes were characterized using natLu-DOTA-erlotinib conjugates. The 177Lu-complexes exhibited high in vitro stability in human serum up to 48 h. They were highly hydrophilic in nature as observed from their log Po/w values (177Lu-DOTA-propyl-Er: -2.5 ± 0.1; 177Lu-DOTA-PEG3-Er: -3.0 ± 0.1; 177Lu-DOTA-PEG6-Er: -3.3 ± 0.1). The MTT assay in A431 human epidermoid carcinoma cell lines indicates that the chemical modification at the terminal alkyne group of the erlotinib molecule does not have significant effect on its TKI property. Biodistribution studies in normal Swiss mice demonstrated fast clearance and excretion of 177Lu-labeled erlotinib complexes. These studies indicate that erlotinib variants with hydrophobic pharmacokinetic modifiers/chelators may enhance the retention of 177Lu-labeled complexes in blood thereby increasing the probability to reach EGFR-expressing tumor.

Validation of a noninvasive aMMP-8 point-of-care diagnostic methodology in COVID-19 patients with periodontal disease.

OBJECTIVES: The aim of this study was to validate an active matrix metalloproteinase (MMP-8) point-of-care diagnostic tool in COVID-19 patients with periodontal disease. SUBJECTS, MATERIALS, AND METHODS: Seventy-two COVID-19-positive and 30 COVID-19-negative subjects were enrolled in the study. Demographic data were recorded, periodontal examination carried out, and chairside tests run for evaluating the expression of active MMP-8 (aMMP-8) in the site with maximum periodontal breakdown via gingival crevicular fluid sampling as well as via a mouth rinse-based kit for general disease activity. In COVID-19-positive patients, the kits were run again once the patients turned COVID-19 negative. RESULTS: The overall (n = 102) sensitivity/specificity of the mouthrinse-based kits to detect periodontal disease was 79.41%/36.76% and that of site-specific kits was 64.71%/55.88% while adjusting for age, gender, and smoking status increased the sensitivity and specificity (82.35%/76.47% and 73.53%/88.24, respectively). Receiver operating characteristic (ROC) analysis for the adjusted model revealed very good area under the ROC curve 0.746-0.869 (p < .001) and 0.740-0.872 (p < .001) (the aMMP-8 mouth rinse and site-specific kits, respectively). No statistically significant difference was observed in the distribution of results of aMMP-8 mouth rinse test (p = .302) and aMMP-8 site-specific test (p = .189) once the subjects recovered from COVID-19. CONCLUSIONS: The findings of the present study support the aMMP-8 point-of-care testing (PoCT) kits as screening tools for periodontitis in COVID-19 patients. The overall screening accuracy can be further increased by utilizing adjunctively risk factors of periodontitis. The reported noninvasive, user-friendly, and objective PoCT diagnostic methodology may provide a way of stratifying risk groups, deciding upon referrals, and in the institution of diligent oral hygiene regimens.

Fluorescence exclusion - a rapid, accurate and powerful method for measuring yeast cell volume.

Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, owing in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from yeast models, but even for these morphologically stereotypical cells, assessment of cell volume has mostly relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle differences in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work highlights how the coupling of FXm with yeast genetics will bring new insights into the complex biology of cell growth.

Spatial transcriptomic and single-nucleus analysis reveals heterogeneity in a gigantic single-celled syncytium.

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

Lineage recording in human cerebral organoids.

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

Is saltwater mouth rinse as effective as chlorhexidine following periodontal surgery?

Design Randomised prospective double-blind study.Case selection Thirty-seven chronic periodontitis patients were randomly prescribed either saltwater (n = 17) or chlorhexidine (n = 20) mouth rinse following open flap debridement. Gingival Index (GI), post-operative pain, mouth rinse satisfaction, matrix metalloproteinase activity and tasting were assessed at baseline, one week and 12 weeks.Data analysis Inter-group and intra-group comparisons were done for all the parameters recorded at different time intervals using Friedman, Wilcoxon signed-rank and Mann-Whitney U tests. P value less than 0.05 was considered statistically significant.Results There was a statistically significant decrease in GI from baseline to week 1/week 12 following surgery in both the groups. However, there was no significant difference between the groups at any time point.Conclusions Saltwater rinses are as efficient as 0.12% chlorhexidine in reducing inflammation following minimal invasive periodontal surgery. Being an inexpensive and easily accessible option, it might be considered the mouth rinse of choice during the early stages of wound healing.

Is There Any Relationship between Poor Menstrual Hygiene Management and Anemia? - A Quantitative Study Among Adolescent Girls of the Urban Slum of Madhya Pradesh.

BACKGROUND: Anemia and poor menstrual hygiene practices have a considerable impact on all aspects of an adolescent girl's life. There is a dire need for evidence on the burden and its association to provide actionable preventive measures. OBJECTIVE: The objective of this study is to explore the magnitude and association between poor Menstrual Hygiene Management (MHM) practices and anemia. MATERIALS AND METHODS: A cross-sectional study conducted among 393 adolescent girls of the urban slum of Bhopal (June 2017-July 2018). Hemoglobin (Hb) was determined by a portable Hb meter (HemoCue). Logistic regression analysis was done to find out if there is any association between poor MHM practices and anemia. RESULTS: The present study found the prevalence of anemia as 38.7% among which 65.1%, 31.5%, and 3.2% were suffering from mild, moderate, and severe forms of anemia, respectively, and 87.3% were practicing poor MHM. Logistic regression found that the MHM, bleeding during menstruation, religion, educational status, and nutritional status of adolescents were significant factors associated with anemia. CONCLUSION: The study concluded that there is a significant association between poor MHM and anemia. Concerted convergent actions focusing on the provision of awareness regarding MHM and sanitary facilities to maintain proper menstrual hygiene are needed to reduce the burden of anemia.

Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages.

The cilium, the sensing centre for the cell, displays an extensive repertoire of receptors for various cell signalling processes. The dynamic nature of ciliary signalling indicates that the ciliary entry of receptors and associated proteins must be regulated and conditional. To understand this process, we studied the ciliary localisation of the odour-receptor coreceptor (Orco), a seven-pass transmembrane protein essential for insect olfaction. Little is known about when and how Orco gets into the cilia. Here, using Drosophila melanogaster, we show that the bulk of Orco selectively enters the cilia on adult olfactory sensory neurons in two discrete, one-hour intervals after eclosion. A conditional loss of heterotrimeric kinesin-2 during this period reduces the electrophysiological response to odours and affects olfactory behaviour. We further show that Orco binds to the C-terminal tail fragments of the heterotrimeric kinesin-2 motor, which is required to transfer Orco from the ciliary base to the outer segment and maintain within an approximately four-micron stretch at the distal portion of the ciliary outer-segment. The Orco transport was not affected by the loss of critical intraflagellar transport components, IFT172/Oseg2 and IFT88/NompB, respectively, during the adult stage. These results highlight a novel developmental regulation of seven-pass transmembrane receptor transport into the cilia and indicate that ciliary signalling is both developmentally and temporally regulated.

Ventral pallidum DRD3 potentiates a pallido-habenular circuit driving accumbal dopamine release and cocaine seeking.

Drugs of abuse induce persistent remodeling of reward circuit function, a process thought to underlie the emergence of drug craving and relapse to drug use. However, how circuit-specific, drug-induced molecular and cellular plasticity can have distributed effects on the mesolimbic dopamine reward system to facilitate relapse to drug use is not fully elucidated. Here, we demonstrate that dopamine receptor D3 (DRD3)-dependent plasticity in the ventral pallidum (VP) drives potentiation of dopamine release in the nucleus accumbens during relapse to cocaine seeking after abstinence. We show that two distinct VP DRD3+ neuronal populations projecting to either the lateral habenula (LHb) or the ventral tegmental area (VTA) display different patterns of activity during drug seeking following abstinence from cocaine self-administration and that selective suppression of elevated activity or DRD3 signaling in the LHb-projecting population reduces drug seeking. Together, our results uncover how circuit-specific DRD3-mediated plasticity contributes to the process of drug relapse.

A meta-analysis on the prevalence of depression in perimenopausal and postmenopausal women in India.

INTRODUCTION: The mental health of perimenopausal and postmenopausal women is a relatively understudied area. This review formally explores the prevalence of depression in perimenopausal and postmenopausal women in India. METHODS: Databases like PubMed, Embase, Cochrane library, Web of Science and Scopus were systematically searched for cross-sectional or cohort studies, providing prevalence of depression in Indian perimenopausal and postmenopausal women. Systematic study selection and data extraction procedures were followed. Quality assessment of individual study was done using AXIS tool. For pooling of effect sizes, the random effects model was used. Funnel plot and Egger's test were used to ascertain publication bias. Subgroup analyses and meta-regression analysis were used to explore heterogeneity in the summary estimates. RESULTS: After a thorough search, ten studies were found to be eligible and included in this review. Pooled estimate for prevalence of depression (random effects model) in perimenopausal and postmenopausal women in India is 42.47 % (95 % CI: 28.73-57.49, I2 = 97.7 %). On visual inspection of the funnel plot and interpreting egger's test (bias: 3.49, SE bias: 3.68, p = 0.37), there was absence of publication bias. CONCLUSION: We documented 42.47 % pooled prevalence of depression in perimenopausal and postmenopausal women in India.