The conventional DNA polymerase machinery is unable to fully replicate the ends of linear chromosomes. To surmount this problem, nearly all eukaryotes use the telomerase enzyme, a specialized reverse ...transcriptase that utizes its own RNA template to add short TG‐rich repeats to chromosome ends, thus reversing their gradual erosion occurring at each round of replication. This unique, non‐DNA templated mode of telomere replication requires a regulatory mechanism to ensure that telomerase acts at telomeres whose TG tracts are too short, but not at those with long tracts, thus maintaining the protective TG repeat ‘cap’ at an appropriate average length. The prevailing notion in the field is that telomere length regulation is brought about through a negative feedback mechanism that ‘counts’ TG repeat‐bound protein complexes to generate a signal that regulates telomerase action. This review summarizes experiments leading up to this model and then focuses on more recent experiments, primarily from yeast, that begin to suggest how this ‘counting’ mechanism might work. The emerging picture is that of a complex interplay between the conventional DNA replication machinery, DNA damage response factors, and a specialized set of proteins that help to recruit and regulate the telomerase enzyme.
The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature ...of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.
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•Rif1 recruits protein phosphatase 1 to telomeres and DNA replication origins•PP1 docking motifs mediate the effect of Rif1 on DNA replication timing•The PP1 recruitment activity of Rif1 counteracts DDK action on Mcm4•Mutations in putative CDK/DDK sites near the PP1 motifs in Rif1 affect PP1 recruitment
The eukaryotic genome is replicated according to a strict temporal program. Here, Bianchi and colleagues find that Rif1, a master regulator of the DNA replication program in yeast and mammals, exerts its effect on DNA replication origins by recruiting protein phosphatase 1 (PP1) to chromosomes. Rif1/PP1 counteracts the positive action of the DDK kinase on the replicative kinase MCM. In a final twist, kinase action on Rif1 is proposed to eventually release PP1 and allow origin firing.
The telomerase enzyme, which synthesizes telomeric DNA repeats, is regulated in
cis at individual chromosome ends by the telomeric protein/DNA complex in a manner dependent on telomere repeat-array ...length. A dynamic interplay between telomerase-inhibiting factors bound at duplex DNA repeats and telomerase-promoting ones bound at single-stranded terminal DNA overhangs appears to modulate telomerase activity and to be directly related to the transient deprotection of telomeres. We discuss recent advances on the mechanism of telomerase regulation at chromosome ends in both yeast and mammalian systems.
One of the most recent innovations in bone augmentation surgery is represented by computer-aided-design/computer-aided-manufacturing (CAD/CAM) customized titanium meshes, which can be used to restore ...vertical bone defects before implant-prosthetic rehabilitations. The aim of this study was to evaluate the effectiveness/reliability of this technique in a consecutive series of cases.
Ten patients in need of bone augmentation before implant therapy were treated using CAD/CAM customized titanium meshes. A digital workflow was adopted to design virtual meshes on 3D bone models. Then, Direct Metal Laser Sintering (DMLS) technology was used to produce the titanium meshes, and vertical ridge augmentation was performed according to an established surgical protocol. Surgical complications, healing complications, vertical bone gain (VBG), planned bone volume (PBV), lacking bone volume (LBV), regenerated bone volume (RBV), average regeneration rate (RR) and implant success rate were evaluated.
All augmented sites were successfully restored with definitive implant-supported fixed partial dentures. Measurements showed an average VBG of 4.5 ± 1.8 mm at surgical re-entry. Surgical and healing complications occurred in 30% and 10% of cases, respectively. Mean values of PBV, LBV, and RBV were 984, 92, and 892 mm
, respectively. The average RR achieved was 89%. All 26 implants were successfully in function after 1 year of follow-up.
The results of this study suggest that the bone augmentation by means of DMLS custom-made titanium meshes can be considered a reliable and effective technique in restoring vertical bone defects.
Tick-borne encephalitis was limited to northeast portions of Italy. We report in Lombardy, a populous region in the northwest, a chamois displaying clinical signs of tickborne encephalitis virus that ...had multiple virus-positive ticks attached, as well as a symptomatic man. Further, we show serologic evidence of viral circulation in the area.
Manufacturing companies operate in a complex environment and need efficient manufacturing processes to remain competitive. Therefore, evaluation methods are essential for decision makers when ...selecting manufacturing innovation projects (MIPs). However, most approaches are not suitable for strategic use and do not consider all relevant evaluation dimensions. To address this issue, this work presents an approach to evaluate and select MIPs holistically, considering potential, effort, and risk. The approach enables the analysis of the strategic impact of an MIP using a fuzzy expert system, and further evaluates the implementation effort and risk using a combination of the Analytical Hierarchy Process and the Exponential Risk Priority Number. The approach was developed using the results of a systematic literature review and expert-based methods. Finally, the approach was validated in an industrial case study and enabled a transparent evaluation of the strategic potential, effort, and risk of two MIPs, leading to informed project selection.
The ability to directly harvest thin and superthin perforator flaps without jeopardizing their vascularity depends on knowledge of the microsurgical vascular anatomy of each perforator within the ...subcutaneous tissue up to the dermis. In this paper, we report our experience with ultrahigh-frequency ultrasound (UHF-US) in the preoperative planning of thin and superthin flaps. Between May 2017 and September 2018, perforators of seven patients were preoperatively evaluated by both ultrasound (using an 18-MHz linear probe) and UHF-US (using 48- and 70-MHz linear probes). Thin flaps (two cases) and superthin flaps (five cases) were elevated for the reconstruction of head and neck oncologic defects and lower limb traumatic defects. The mean flap size was 6.5×15 cm (range, 5×8 to 7.5×23 cm). No complications occurred, and all flaps survived completely. In all cases, we found 100% agreement between the preoperative UHF-US results and the intraoperative findings. The final reconstructive outcomes were considered satisfactory by both the surgeon and the patients. In conclusion, UHF-US was found to be very useful in the preoperative planning of thin and superthin free flaps, as it allows precise anticipation of very superficial microvascular anatomy. UHF-US may represent the next frontier in thin, superthin, and pure skin perforator flap design.
Fresh (frozen/thawed) muscle samples from four 2–12-year-old roe deer (
Capreolus capreolus
) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about ...180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome
c
oxidase subunit I gene (
cox1
) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5–3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2–3 mm long, with hair-like protrusions. Sequencing of
cox1
revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to
Sarcocystis gracilis
(
n
= 24) or to a
Sarcocystis taeniata
-like species (
n
= 19), whereas those with finger- and hair-like protrusions belonged to
Sarcocystis silva
(
n
= 27) and
Sarcocystis capreolicanis
(
n
= 7), respectively. The 19
cox1
sequences of the
S. taeniata
-like species, comprising five haplotypes, differed from each other at 0–16 of 1038 nucleotide positions (98.5–100% identity). They differed from 25 previous
cox1
sequences of
S. taeniata
from moose and sika deer (with 98.0–100% intraspecific identity), at 33–43 nucleotide positions (95.9–96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on
cox1
sequences, the two populations formed two separate monophyletic clusters. The
S. taeniata
-like species in roe deer was therefore considered to represent a separate species, which was named
Sarcocystis linearis
n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of
S. linearis
(98.1–99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of
S. taeniata
. The new 18S rRNA gene sequences of
S. linearis
shared an identity of 97.9–99.6% with those of
S. taeniata
(overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of
S. linearis
were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8–3.2 μm long, 0.3–0.4 μm wide and about 0.02–0.03 μm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3–0.4 μm wide and 0.7–0.9 μm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of
S. linearis
was consistent with that of an unnamed
Sarcocystis
sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of
S. gracilis
and
S. silva
were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in
S. gracilis
(as seen in previous SEM studies) and revealing tightly packed, erect 6–7-μm-long villus-like protrusions having regularly distributed round depressions on their surface in
S. silva
. The sequencing of
cox1
of 7, 24 and 27 new isolates of
S. capreolicanis
,
S. gracilis
and
S. silva
, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of
S. capreolicanis
and
S. silva
than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of
S. gracilis
.
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