Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.     

Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma

Approximately 1%–2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next‐generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four‐case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.     

Simultaneous analysis of lysine, Nepsilon-carboxymethyllysine and lysinoalanine from proteins

Protein quality was assayed by simultaneous measurement of lysine (Lys), carboxymethyllysine (CML) and lysinoalanine (LAL). GC-FID analysis of N-tert-butyl dimethylsilyl (tBDMSi) derivatives of these amino acids was undertaken. tBDMSi derivates were separated on a CP-SIL 5CB commercially fused silica capillary column (25 m x 0.25 mm i.d., 0.25 microm film thickness) employing a thermal gradient programmed from 200 to 300 degrees C. The identity of tBDMSi derivatives of Lys, CML and LAL was established by GC-MS while FID detection was employed for quantification. Analytical parameters such as linearity (lysine 350-4200 microM, LAL 3-81 microM, CML 16-172 microM), precision (1-13% variation coefficients), accuracy (85-108% average recovery) and limits of detection (lysine 0.4 mg/100 g protein, LAL 5.0 mg/100 g protein, CML 3.4 mg/100 g protein) and quantification (lysine 1.4 mg/100g protein, LAL 15.2 mg/100 g protein, CML 11.2 mg/100 g protein) were determined for validation of the analytical approach. Model systems and real foods have been studied. Kinetic of CML formation from different food proteins (BSA, soy protein, casein and gluten) was performed employing model systems. Carboxymethylation rate depended on the source of protein. Maillard reaction progressed to advanced stages damaging the protein quality of stored infant foods, soy drinks, boiled eggs and dry powdered crepes. CML values ranged from 62 to 440 mg/100 g protein were measured. LAL was also formed during boiling eggs (21-68 mg/100g protein) indicating additional damage by crosslinking reaction. In agreement, lysine content was affected by both food processing and storage.     

Widespread colonisation of Tanzanian catchments by introduced Oreochromis tilapia fishes: the legacy from decades of deliberate introduction

From the 1950s onwards, programmes to promote aquaculture and improve capture fisheries in East Africa have relied heavily on the promise held by introduced species. In Tanzania these introductions have been poorly documented. Here we report the findings of surveys of inland water bodies across Tanzania between 2011 and 2017 that clarify distributions of tilapiine cichlids of the genus Oreochromis. We identified Oreochromis from 123 sampling locations, including 14 taxa restricted to their native range and three species that have established populations beyond their native range. Of these three species, the only exotic species found was blue-spotted tilapia (Oreochromis leucostictus), while Nile tilapia (Oreochromis niloticus) and Singida tilapia (Oreochromis esculentus), which are both naturally found within the country of Tanzania, have been translocated beyond their native range. Using our records, we developed models of suitable habitat for the introduced species based on recent (1960–1990) and projected (2050, 2070) East African climate. These models indicated that presence of suitable habitat for these introduced species will persist and potentially expand across the region. The clarification of distributions provided here can help inform the monitoring and management of biodiversity, and inform policy related to the future role of introduced species in fisheries and aquaculture.

     

Bundle sheath extensions affect leaf structural and physiological plasticity in response to irradiance

Coordination between structural and physiological traits is key to plants' responses to environmental fluctuations. In heterobaric leaves, bundle sheath extensions (BSEs) increase photosynthetic performance (light‐saturated rates of photosynthesis, A_max) and water transport capacity (leaf hydraulic conductance, K_leaf). However, it is not clear how BSEs affect these and other leaf developmental and physiological parameters in response to environmental conditions. The obscuravenosa (obv) mutation, found in many commercial tomato varieties, leads to absence of BSEs. We examined structural and physiological traits of tomato heterobaric and homobaric (obv) near‐isogenic lines grown at two different irradiance levels. K_leaf, minor vein density, and stomatal pore area index decreased with shading in heterobaric but not in homobaric leaves, which show similarly lower values in both conditions. Homobaric plants, on the other hand, showed increased A_max, leaf intercellular air spaces, and mesophyll surface area exposed to intercellular airspace (S_mes) in comparison with heterobaric plants when both were grown in the shade. BSEs further affected carbon isotope discrimination, a proxy for long‐term water‐use efficiency. BSEs confer plasticity in traits related to leaf structure and function in response to irradiance levels and might act as a hub integrating leaf structure, photosynthetic function, and water supply and demand.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP(ow) (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of > or =2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP(ow) value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP(ow) around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP(ow) of aliphatic alcohols correlates with their toxic effects, as octanol (logP(ow) = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP(ow) = 3.4) or 1-decanol (logP(ow) = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP(ow) = 3.2) into 3-methylcatechol (logP(ow) = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.     

Fine needle aspiration cytology of neurothekeoma. A case report

BACKGROUND: Neurothekeoma (NT) is a rare, benign neoplasm of soft parts with a distinctive histologic appearance. To our knowledge, the cytologic findings have not been described before. We present a case of NT with the cytologic features on fine needle aspiration cytology (FNAC). CASE: A 54-year-old female presented with a circumscribed nodule in the left breast. The lesion was evaluated by FNAC. The smears showed an abundant, metachromatic, myxoid matrix with fusiform and epithelioid cells, some binucleated or multinucleated, loose or in groups and sometimes forming concentric whorls. The lesion was removed, and the diagnosis of NT was made after histopathologic study. CONCLUSION: NT is an extremely rare neoplasm in the mammary region. Fusiform and epithelioid cells arranged in concentric whorls in a myxoid tumor of soft tissue are a distinctive characteristic of this neoplasm and can suggest the diagnosis.