Error-free bypass of 2-hydroxyadenine by human DNA polymerase λ with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-

Error-free bypass of 2-hydroxyadenine by human DNA polymerase λ with Proliferating Cell Nuclear Antigen and Replication Protein A in different sequence contexts

1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol λ, to bypass the 2-OH-A lesion. DNA pol λ can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol λ was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol λ active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol λ in combination with PCNA and RP-A

Experimental Tests on Cement Mortars Manufactured with Hemp Flour

Aim: Currently, research and market needs are aimed at the production of more and more specific mortars and concrete with the best properties and mix design directed towards increasingly specific uses. Background: This research starts with a discussion on cement and aggregate assortments, which are the basic properties of mortars. Objective: The choice of suitable additives able to correct the rheological, workability and resistance properties of the mixture is focused. Methods: In this framework, the current research activity involves some experimental laboratory tests on cement mortars manufactured with hemp flour, intended as a “natural” additive. The aim is the search for any improvements given by the hemp flour to the physical and/or mechanical performances of the tested mortars. Results: Hemp flour, due to bonding properties, increases the compactness, and therefore, the resistance to environmental factors by the examined cement mortars. The rheological features of mixtures with different percentages of hemp flour, whose maximum water percentage absorbed was resulted to be 125%, were investigated. It was found that standardised sand reduces shrinkage phenomena of tested specimens. Moreover, the compression and bending moment behaviour of manufactured specimens was analysed with the aim of finding the optimal weight percentage of hemp flour to improve their mechanical performances. Conclusion: The experimentation performed leads to several conclusions and interesting future developments. It was found that 3% is the best-weighted percentage of hemp flour to increase the mechanical properties of examined mortars made of standardised sand. As further developments, there is a need to identify the limit percentage of hemp flour to be added to the mixture, so as to avoid the decay of the mechanical properties. In addition, further experiments must be carried out on concretes, where the presence of gravel leads to improve the stability and mechanical resistance of the cement matrix

Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

‘Classical' non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instabilit

Nanoroughness, Surface Chemistry and Drug Delivery Control by Atmospheric Plasma Jet on Implantable Devices

Implantable devices need specific tailored surface morphologies and chemistries to interact with the living systems or to actively induce a biological response also by the release of drugs or proteins. These customised requirements foster technologies that can be implemented in additive manufacturing systems. Here we present a novel approach based on spraying processes that allows to control separately topographic features in the submicron range ( 3d 60 nm - 2 \ub5m), ammine or carboxylic chemistry and fluorophore release even on temperature sensitive biodegradable polymers such as polycaprolactone (PCL). We developed a two-steps process with a first deposition of 220 nm silica and poly(lactic-co-glycolide) (PLGA) fluorescent nanoparticles by aerosol followed by the deposition of a fixing layer by atmospheric pressure plasma jet (APPJ). The nanoparticles can be used to create the nano-roughness and to include active molecule release, while the capping layer ensures stability and the chemical functionalities. The process is enabled by a novel APPJ which allows deposition rates of 10 - 20 nm\ub7s-1 at temperatures lower than 50 \ub0C using argon as process gas. This approach was assessed on titanium alloys for dental implants and on PCL films. The surfaces were characterized by FT-IR, AFM and SEM. Titanium alloys were tested with pre-osteoblasts murine cells line, while PCL film with fibroblasts. Cell behaviour was evaluated by viability and adhesion assays, protein adsorption, cell proliferation, focal adhesion formation and SEM. The release of a fluorophore molecule was assessed in the cell growing media, simulating a drug release. Osteoblast adhesion on the plasma treated materials increased by 20% with respect to commercial titanium alloys implants. Fibroblast adhesion increased by a 100% compared to smooth PCL substrate. The release of the fluorophore by the dissolution of the PLGA nanoparticles was verified and the integrity of the encapsulated drug model confirmed

Repair of a spinal pseudomeningocele in a delayed postsurgical cerebrospinal fluid leak using titanium U-clips: Technical note

Introduction: Chronic cerebrospinal fluid leak (CCFL) represents a possible event following spine surgery, with an incidence rate ranging from 5 to 18%. In literature have been reported few modification techniques that involved the use of titanium U-clips with the aim to reduce dural traumatism during its closure but only after non-accidental durotomy. Case illustration: We report the case of a 47-year-old female with history of L5-S1 microsurgical discectomy. After one year the patient was admitted to our unit because she presented progressive skin swelling in the lumbar region, intermittent headache and recurrent episodes of lipothymia. One-year MRI showed a voluminous subcutaneous cerebrospinal fluid (CSF)-like intensity collection in the T2-weighted sequences and communicating with the right L5-S1 interlaminar space. A second surgery revealed pseudomeningocele and a 5 mm dural defect is identified. At first, a tobacco pouch was created, and a suture with Prolene 6–0, a non-absorbable, synthetic monofilament, is made on the previously dissected tissue. In a second step, 2 medium/short non-penetrating titanium U-clips are applied with a multi-clip applier. There was no sign of recurrent pseudomeningocele in the 2-year follow-up. Conclusion: The authors presented a novel use of titanium U-clips to manage an insidious and complex case of pseudomeningocele in a CCFL. U-clips represent a safe and effective tool in CCFL repair

Rubber hand illusion induced by touching the face ipsilaterally to a deprived hand: evidence for plastic "somatotopic" remapping in tetraplegics

Background: Studies in animals and humans indicate that the interruption of body-brain connections following spinal cord injury (SCI) leads to plastic cerebral reorganization.Objective: To explore whether inducing the Rubber Hand Illusion (RHI) via synchronous multisensory visuo-tactile bodily stimulation may reveal any perceptual correlates of plastic remapping in SCI.Methods: In 16 paraplegic, 16 tetraplegic and 16 healthy participants we explored whether RHI may be induced by tactile stimuli involving not only the left hand but also the left hemi-face. Touching the participants actual hand or face was either synchronous or asynchronous with tactile stimuli seen on a rubber hand. We assessed two components of the illusion, namely perceived changes in the real hand in space (indexed by proprioceptive drift) and ownership of the rubber hand (indexed by subjective responses to an ad-hoc questionnaire).Results: Proprioceptive drift and ownership were found in the healthy group only in the condition where the left real and fake hand were touched simultaneously. In contrast, no drift was found in the SCI patients who, however, showed ownership after both synchronous and asynchronous hand stroking. Importantly, only tetraplegics showed the effect also after synchronous face stroking.Conclusions: RHI may reveal plastic phenomena in SCI. In hand representation-deprived tetraplegics, stimuli on the face (represented contiguously in the somatic and motor systems), drive the sense of hand ownership. This hand-face remapping phenomenon may be useful for restoring a sense of self in massively deprived individuals

Physical–chemical properties of biogenic selenium nanostructures produced by Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite (www.frontiersin.org) was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by www.frontiersin.org upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02-G_e and SeNPsSeITE02-P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416–640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes

Correlation between sperm parameters and circulating thyroid hormones and testosterone concentrations in Labrador Retriever dog

Thyroid hormones physiologically regulate the semen quality, by altering testosterone concentrations, and different seminal parameters, as well as sperm motility, viability and semen volume. Serum thyroxine (T4), free thyroxine (fT4), thyroid-stimulating hormone (TSH) and testosterone (T) concentrations were measured in 26 adult Labrador Retriever dogs (n. 20 normospermic and n. 6 azoospermic groups), aged 3 ± 0.5 years old, to determine their specific references, by taking into account the related conventional and kinematic sperm parameters and clinical ultrasound evaluations. The mean values of T4, fT4, TSH and T concentrations, as soon as those of sperm parameters and clinical evaluations of normospermic group were in line with dog's physiological range of literature data. Normospermic group showed significant correlations between T4 and T (r = −0.681; p < .01), sperm progressive motility (%) (r = −0.623; p < .01), and sperm non-progressive motility (%) (r = 0.625; p < .02). The azoospermic group showed higher T4, fT4, TSH and lower T concentrations, compared to normospermic group, and a positive correlation between T4 and T (r = 0.8548; p < .046). The use of breed-specific hormonal ranges and sperm parameters will improve the knowledge of their interaction in Labrador Retriever dogs, adding a new segment of scientific literature.Highlights Thyroxine (T4), free thyroxine (fT4), thyroid stimulating hormone (TSH) and testosterone (T) concentrations were measured in male dogs. Conventional and kinematic sperm parameters were also evaluated. Dogs were divided in 20 normospermic and n. 6 azoospermic Labrador Retrivers. Azoospermic group showed higher values of T4, fT4, TSH and lower values of T compared to normospermic Use of breed-specific hormonal ranges and sperm parameters will improve the knowledge of scientific literature