A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFß deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.

Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but is still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development (referred to as the RAPID-E protocol). Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II, but additionally expressed Collagen X, indicative of hypertrophy. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs.

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

Catalytic Alkyne Semihydrogenation with Polyhydride Ni/Ga Clusters.

The bimetallic, decanuclear Ni3 Ga7 -cluster of the formula [Ni3 (GaTMP)3 (µ2 -GaTMP)3 (µ3 -GaTMP)] (1, TMP=2,2,6,6-tetramethylpiperidinyl) reacts reversibly with dihydrogen under the formation of a series of (poly-)hydride clusters 2. Low-temperature 2D NMR experiments at -80 °C show that 2 consist of a mixture of a di- (2Di ), tetra- (2Tetra ) and hexahydride species (2Hexa ). The structures of 2Di and 2Tetra are assessed by a combination of 2D NMR spectroscopy and DFT calculations. The cooperation of both metals is essential for the high hydrogen uptake of the cluster. Polyhydrides 2 are catalytically active in the semihydrogenation of 4-octyne to 4-octene with good selectivity. The example is the first of its kind and conceptually relates properties of molecular, atom-precise transition metal/main group metal clusters to the respective solid-state phase in catalysis.

Directed differentiation of hPSCs through a simplified lateral plate mesoderm protocol for generation of articular cartilage progenitors.

Developmentally, the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced both LP derived prechondrocytes and preosteoblasts from human pluripotent stem cells (hPSC) through a common progenitor in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocyte- and preosteoblast-like cells can be generated. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to investigate further cartilage development and in the development of joint cartilage for potential treatments.

Pluripotent stem cells for skeletal tissue engineering.

Here, we review the use of human pluripotent stem cells for skeletal tissue engineering. A number of approaches have been used for generating cartilage and bone from both human embryonic stem cells and induced pluripotent stem cells. These range from protocols relying on intrinsic cell interactions and signals from co-cultured cells to those attempting to recapitulate the series of steps occurring during mammalian skeletal development. The importance of generating authentic tissues rather than just differentiated cells is emphasized and enabling technologies for doing this are reported. We also review the different methods for characterization of skeletal cells and constructs at the tissue and single-cell level, and indicate newer resources not yet fully utilized in this field. There have been many challenges in this research area but the technologies to overcome these are beginning to appear, often adopted from related fields. This makes it more likely that cost-effective and efficacious human pluripotent stem cell-engineered constructs may become available for skeletal repair in the near future.

Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone.

Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.

Mast Cell Cytokines IL-1, IL-33, and IL-36 Mediate Skin Inflammation in Psoriasis: A Novel Therapeutic Approach with the Anti-Inflammatory Cytokines IL-37, IL-38, and IL-1Ra.

Psoriasis (PS) is a skin disease with autoimmune features mediated by immune cells, which typically presents inflammatory erythematous plaques, and is associated with many comorbidities. PS exhibits excessive keratinocyte proliferation, and a high number of immune cells, including macrophages, neutrophils, Th1 and Th17 lymphocytes, and mast cells (MCs). MCs are of hematopoietic origin, derived from bone marrow cells, which migrate, mature, and reside in vascularized tissues. They can be activated by antigen-provoking overexpression of proinflammatory cytokines, and release a number of mediators including interleukin (IL)-1 and IL-33. IL-1, released by activated keratinocytes and MCs, stimulates skin macrophages to release IL-36-a powerful proinflammatory IL-1 family member. IL-36 mediates both innate and adaptive immunity, including chronic proinflammatory diseases such as psoriasis. Suppression of IL-36 could result in a dramatic improvement in the treatment of psoriasis. IL-36 is inhibited by IL-36Ra, which binds to IL-36 receptor ligands, but suppression can also occur by binding IL-38 to the IL-36 receptor (IL-36R). IL-38 specifically binds only to IL-36R, and inhibits human mononuclear cells stimulated with IL-36 in vitro, sharing the effect with IL-36Ra. Here, we report that inflammation in psoriasis is mediated by IL-1 generated by MCs-a process that activates macrophages to secrete proinflammatory IL-36 inhibited by IL-38. IL-37 belongs to the IL-1 family, and broadly suppresses innate inflammation via IL-1 inhibition. IL-37, in murine models of inflammatory arthritis, causes the suppression of joint inflammation through the inhibition of IL-1. Therefore, it is pertinent to think that IL-37 can play an inhibitory role in inflammatory psoriasis. In this article, we confirm that IL-38 and IL-37 cytokines emerge as inhibitors of inflammation in psoriasis, and hold promise as an innovative therapeutic tool.

The use of scanning electron microscopy and energy dispersive X-ray spectroscopy in a case of occupational death.

Scanning electron microscopy is a technique that provides high-resolution images at the micro- and nano-scale. The combination of scanning electron microscopy and energy dispersive X-ray spectroscopy analysis is developing fast for application in forensic science. In this work, we report a case of work-related traumatic death of a 50-year-old man. The autopsy showed cranial fractures with cerebral haemorrhage. It was more difficult to understand the accident dynamics because the body had been shifted from the accident site to mask what had really taken place. Scanning electron microscopy/energy dispersive X-ray spectroscopy was used to identify the material of the impacting tool and to establish the possible legal responsibility of the employer. In this study, we demonstrate that scanning electron microscopy/energy dispersive X-ray spectroscopy is a useful forensic tool for the analysis of biological samples. Further, for studying the lacerations on the corpse from doubtful blunt tools, scanning electron microscopy/energy dispersive X-ray spectroscopy can assist in demonstrating that the scene has been falsified, as it was in this case.

Relating the rate of growth of metal nanoparticles to cluster size distribution in electroless deposition.

Electroless deposition on patterned silicon substrates enables the formation of metal nanomaterials with tight control over their size and shape. In the technique, metal ions are transported by diffusion from a solution to the active sites of an autocatalytic substrate where they are reduced as metals upon contact. Here, using diffusion limited aggregation models and numerical simulations, we derived relationships that correlate the cluster size distribution to the total mass of deposited particles. We found that the ratio ξ between the rates of growth of two different metals depends on the ratio γ between the rates of growth of clusters formed by those metals through the linearity law ξ = 14(γ - 1). We then validated the model using experiments. Different from other methods, the model derives k using as input the geometry of metal nanoparticle clusters, decoded by SEM or AFM images of samples, and a known reference.

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

Foot and Soccer Referees': A Pilot Study Searching "Performance" Throughout Prevention.

Soccer refereeing is a "not-conventional" sport in which aerobic workload is prevalent. Along the years, several studies have attempted to define best markers of referees' performance. Many studies focused their attention on field tests and their relationship with aerobic power. Instead, in this study, starting by a medical assessment satisfying the FIFA 11+ criteria for injuries prevention, we have investigated the foot of soccer referees and we have also wanted to find possible and/or unexpected improvements in performance. As performance marker, we have used the referral field test for soccer referees that is internationally validated and known as Yo-Yo test (YYiR1). While standardized foot posture index (FPI) questionnaire was used for screening foot referees conditions (40 young, all men by sex, with mean age 23.47 ± 4.36). Analyzing collected data, we have demonstrated by means of Read-Cressie Chi square test that neutral FPI is an important favor item affecting YYiR1 results. Further studies will be necessary in order to confirm our pilot investigation.

Antibiotic sensitivities of coagulase-negative staphylococci and Staphylococcus aureus in hip and knee periprosthetic joint infections: does this differ if patients meet the International Consensus Meeting Criteria?

INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the main pathogens responsible for prosthetic joint infections (PJIs). As normal inhabitants of human skin, it is often difficult to define if they are contaminants, or if they have an active role in initiating infection. This study aims to evaluate differences in CoNS organisms (Staphylococcus hominis, Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus warneri) and Staphylococcus aureus in terms of isolation rate and antimicrobial susceptibility from patients who met the International Consensus Meeting (ICM) criteria for PJIs and those who did not. METHODS: Staphylococci isolates from January 2014 to December 2015 retrieved from patients undergoing revision joint arthroplasty were classified in accordance with criteria established by the ICM of Philadelphia. RESULTS: As per the consensus classification, 50 CoNS and 39 S. aureus infections were recognized as pathogens, while 16 CoNS and four S. aureus were considered as contaminants. Frequency of isolation of S. aureus was significantly higher in infected patients than in those without infection, while no significant differences were observed among CoNS. Resistance to levofloxacin, erythromycin, gentamicin trimethoprim/sulfamethoxazole, and rifampicin was significantly more frequent in S. haemolyticus than in the other species, as well as resistance to erythromycin and gentamicin in S. hominis. In comparison to S. aureus, CoNS were significantly more resistant to daptomycin and gentamicin and more susceptible to rifampicin. CONCLUSION: CoNS, other than Staphylococcus epidermidis, are frequently isolated from PJIs, and their infective role and antimicrobial susceptibility need to be assessed on an individual patient basis. S. haemolyticus seems to emerge as responsible for PJI in a large volume of patients, and its role needs to be further investigated, also considering its pattern of resistance.

Silica diatom shells tailored with Au nanoparticles enable sensitive analysis of molecules for biological, safety and environment applications.

Diatom shells are a natural, theoretically unlimited material composed of silicon dioxide, with regular patterns of pores penetrating through their surface. For their characteristics, diatom shells show promise to be used as low cost, highly efficient drug carriers, sensor devices or other micro-devices. Here, we demonstrate diatom shells functionalized with gold nanoparticles for the harvesting and detection of biological analytes (bovine serum albumin-BSA) and chemical pollutants (mineral oil) in low abundance ranges, for applications in bioengineering, medicine, safety, and pollution monitoring.

An Overview of Lipid Droplets in Cancer and Cancer Stem Cells.

For decades, lipid droplets have been considered as the main cellular organelles involved in the fat storage, because of their lipid composition. However, in recent years, some new and totally unexpected roles have been discovered for them: (i) they are active sites for synthesis and storage of inflammatory mediators, and (ii) they are key players in cancer cells and tissues, especially in cancer stem cells. In this review, we summarize the main concepts related to the lipid droplet structure and function and their involvement in inflammatory and cancer processes.

Nano-topography Enhances Communication in Neural Cells Networks.

Neural cells are the smallest building blocks of the central and peripheral nervous systems. Information in neural networks and cell-substrate interactions have been heretofore studied separately. Understanding whether surface nano-topography can direct nerve cells assembly into computational efficient networks may provide new tools and criteria for tissue engineering and regenerative medicine. In this work, we used information theory approaches and functional multi calcium imaging (fMCI) techniques to examine how information flows in neural networks cultured on surfaces with controlled topography. We found that substrate roughness S a affects networks topology. In the low nano-meter range, S a = 0-30 nm, information increases with S a . Moreover, we found that energy density of a network of cells correlates to the topology of that network. This reinforces the view that information, energy and surface nano-topography are tightly inter-connected and should not be neglected when studying cell-cell interaction in neural tissue repair and regeneration.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Smart greenhouse fuzzy logic based control system enhanced with wireless data monitoring.

Greenhouse climate control is complicated procedure since the number of variables involved on it and which are dependent on each other. This paper presents a contribution to integrate greenhouse inside climate key's parameters, leading to promote a comfortable micro-climate for the plants growth while saving energy and water resources. A smart fuzzy logic based control system was introduced and improved through specific measure to the temperature and humidity correlation. As well, the system control was enhanced with wireless data monitoring platform for data routing and logging, which provides real time data access. The proposed control system was experimentally validated. The efficiency of the system was evaluated showing important energy and water saving.

Nanoscale reduction of graphene oxide thin films and its characterization.

In this paper, we report on a method to reduce thin films of graphene oxide (GO) to a spatial resolution better than 100 nm over several tens of micrometers by means of an electrochemical scanning probe based lithography. In situ tip-current measurements show that an edged drop in electrical resistance characterizes the reduced areas, and that the reduction process is, to a good approximation, proportional to the applied bias between the onset voltage and the saturation thresholds. An atomic force microscope (AFM) quantifies the drop of the surface height for the reduced profile due to the loss of oxygen. Complementarily, lateral force microscopy reveals a homogeneous friction coefficient of the reduced regions that is remarkably lower than that of native graphene oxide, confirming a chemical change in the patterned region. Micro Raman spectroscopy, which provides access to insights into the chemical process, allows one to quantify the restoration and de-oxidation of the graphitic network driven by the electrochemical reduction and to determine characteristic length scales. It also confirms the homogeneity of the process over wide areas. The results shown were obtained from accurate analysis of the shift, intensity and width of Raman peaks for the main vibrational bands of GO and reduced graphene oxide (rGO) mapped over large areas. Concerning multilayered GO thin films obtained by drop-casting we have demonstrated an unprecedented lateral resolution in ambient conditions as well as an improved control, characterization and understanding of the reduction process occurring in GO randomly folded multilayers, useful for large-scale processing of graphene-based material.

Clinical characteristics of chronic kidney disease of non-traditional causes in women of agricultural communities in El Salvador.

INTRODUCTION: A chronic kidney disease of non-traditional causes (CKDu) has emerged in Central America and elsewhere, predominantly affecting male farmworkers. In El Salvador (2009), it was the second cause of death in men > 18 years old. Causality has not been determined. Most available research focused on men and there is scarce data on women. OBJECTIVES: Describe the clinical and histopathologic characteristics of CKDu in women of agricultural communities in El Salvador. METHODOLOGY: A descriptive study was carried out in 10 women with CKDu stages 2, 3a, and 3b. Researchers studied demographics, clinical examination; hematological and biochemical analyses, urine sediment, renal injury markers, and assessed renal, cardiac, and peripheral arteries, liver, pancreas, and lung anatomy and functions. Kidney biopsy was performed in all. Data was collected on the Lime Survey platform and exported to SPSS 19.0. RESULTS: Patient distribution by stages: 2 (70%), 3a (10%), 3b (20%). Occupation: agricultural 7; non-agricultural 3. RISK FACTORS: agrochemical exposure 100%; farmworkers 70%; incidental malaria 50%, NSAIDs use 40%; hypertension 40%. SYMPTOMS: nocturia 50%; dysuria 50%; arthralgia 70%; asthenia 50%; cramps 30%, profuse sweating 20%. Renal markers: albumin creatinine ratio (ACR) > 300 mg/g 90%; ß microglobulin and neutrophil gelatinase- associated lipocalin (NGAL) presence in 40%. Kidney function: hypermagnesuria 100%; hyperphosphaturia 50%, hypercalciuria 40%; hypernatriuria 30%; hyponatremia 60%, hypocalcemia 50%. Doppler: tibial artery damage 40%. Neurological: reflex abnormalities 30%; Babinski and myoclonus 20%. Neurosensorial hypoacusis 70%. Histopathology: damage restricted mostly to the tubulo-interstitium, urine was essentially bland. CONCLUSIONS: CKDu in women is a chronic tubulointerstitial nephropathy with varied extrarenal symptoms.