Direct effects of Ca2+/calmodulin on actin filament formation.

Actin filament formation plays a pivotal role in the development, regeneration and modulation of the morphologies and physiological functions of subcellular compartments and entire cells. All of these processes require tight temporal and spatial control of F-actin assembly. Recent work has shed new light on the control of actin filament formation by Ca2+ as very fast, transient messenger allowing for defined responses to signal intensities spanning several orders of magnitude. Recent discoveries highlight that a small but rapidly growing set of actin nucleators and related proteins, i.e. factors that have the power to promote the formation of new actin filaments in cells, are tightly controlled by the Ca2+ sensor protein CaM. We here review the cellular functions and the molecular mechanisms that couple Ca2+ signaling to the cytoskeletal functions of these factors. This set of proteins currently includes one actin nucleator of the formin family (INF2), the WH2 domain-based actin nucleator Cobl and its ancestor protein Cobl-like as well as fesselin/synaptopodin-2/myopodin and myelin basic protein (MBP). Considering the mechanistic principles of Ca2+ control of actin filament formation unveiled thus far and the diverse cell biological processes involving Ca2+ signaling it is obvious that our understanding of the cell biological crosstalk of Ca2+ transients with the in part highly specialized actin cytoskeletal structures observed in different cell types is only at its infancy.

The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin.

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer-binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl's functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl's actin binding properties and furthermore promotes Cobl's previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl's cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells-a key process in neuronal network formation.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Association between serum biochemical levels, related to bone metabolism and Parkinson's disease.

BACKGROUND: Vitamin D insufficiency and serum calcium disturbance have been reported to be more common in Parkinson's disease (PD) patients than in healthy control subjects, which may be due to a chronic disease or reduced mobility contributes to these relatively disturbances. Because of the high-vitamin D insufficiency in our population, we aimed to compare a biochemical levels which are related to bone metabolism, in PD patients in comparison with age-matched healthy controls, for the 1(st) time in a Middle East population. MATERIALS AND METHODS: This case-control study was involved 105 (20 were excluded) PD patients, who were age- and -sex matched with 112 controls. 25-hydroxyvitamin D (25OHD) and parathyroid hormone analyzed by enzyme immunoassay; another laboratory data including, calcium, phosphorous, and alkaline phosphatase were performed by spectrophotometric methods. RESULTS: There was no significant difference in 25OHD between PD patients and control group (P = 0.071). 25OHD level was not significantly different in PD patients compared to controls {odds ratio 1.003, (confidence interval [CI], 0.98-1.02), P value 0.793}. None of the other biochemical levels did not induce more chance for PD, only we observed in men has more risk of PD than women (odds ratio 2.53, [CI, 1.27-5.03], P value 0.008). CONCLUSION: Our data do not support a possible role of vitamin D insufficiency in PD. Regarding to variable changes in biochemical markers in PD patients than in controls; further studies are suggested to determine any plausibility role of them as a causal relationship or as an outcome of PD.

Membrane shapers from two distinct superfamilies cooperate in the development of neuronal morphology.

Membrane-shaping proteins are driving forces behind establishment of proper cell morphology and function. Yet, their reported structural and in vitro properties are noticeably inconsistent with many physiological membrane topology requirements. We demonstrate that dendritic arborization of neurons is powered by physically coordinated shaping mechanisms elicited by members of two distinct classes of membrane shapers: the F-BAR protein syndapin I and the N-Ank superfamily protein ankycorbin. Strikingly, membrane-tubulating activities by syndapin I, which would be detrimental during dendritic branching, were suppressed by ankycorbin. Ankycorbin's integration into syndapin I-decorated membrane surfaces instead promoted curvatures and topologies reflecting those observed physiologically. In line with the functional importance of this mechanism, ankycorbin- and syndapin I-mediated functions in dendritic arborization mutually depend on each other and on a surprisingly specific interface mediating complex formation of the two membrane shapers. These striking results uncovered cooperative and interdependent functions of members of two fundamentally different membrane shaper superfamilies as a previously unknown, pivotal principle in neuronal shape development.

A comparative investigation into relative bond strengths of Damon3, Damon3MX, and APC II brackets using different primer and adhesive combinations.

This investigation measured and compared shear bond strength (SBS) and adhesive remnant indices (ARIs) of Damon3 and Damon3MX brackets bonded with their recommended primer/adhesive combination or Transbond XT primer/adhesive, with APC II brackets bonded using Transbond XT. Sixty non-carious human third molars were collected and randomly divided into six equal groups of 10. Amongst these, one group was used to standardize the testing methodology, with the remainder constituting the five experimental groups. Upper right central incisor brackets represented each bracket type. Specifically, Damon3 brackets were bonded using either OrthoSolo primer/Blugloo (recommended) or Transbond XT primer/adhesive; Damon3MX brackets were bonded using OrthoSolo primer/Grengloo (recommended) or Transbond XT primer/adhesive, and APC II brackets were bonded with Transbond XT primer. Brackets were debonded by shear force using an Instron machine and the SBS measured. Scores for ARI were determined for all groups after bracket failure by magnified inspection of the tooth surface. Logrank tests showed a significantly higher SBS with Damon3 brackets bonded with OrthoSolo/Blugloo compared with Transbond XT, but no significant differences between the SBS of Damon3MX brackets bonded with OrthoSolo/Grengloo compared with Transbond XT. There were no significant differences in SBS of all three bracket types when bonded with Transbond XT. Pearson's chi-square test showed no difference in the locus of debond. All three adhesive systems are reliable when bonding Damon3 and Damon3MX brackets. Some caution should be taken when using Damon3 brackets bonded with OrthoSolo/Blugloo due to the higher SBS, although no enamel fractures were noted in this study.

Pain Control After Mastectomy in Transgender Patients: Ultrasound-guided Pectoral Nerve Block II Versus Conventional Intercostal Nerve Block: A Randomized Clinical Trial.

BACKGROUND: Mastectomy is sometimes performed in transgender patients, which may damage the regional nerves such as the pectoral and intercostobrachial nerves, leading to postoperative pain. An ultrasound-guided nerve block can be used to track and block the nerves properly. OBJECTIVES: This study aimed to compare the ultrasound-guided type-II pectoral nerve block with the blind (conventional) intercostal nerve block (ICNB) for pain control after breast tissue reconstruction surgery in transgender patients. METHODS: In the present single-blind randomized clinical trial, 47 patients were randomly divided into two groups: (A) Ultrasound-guided type-II pectoral nerve block (n = 23) and (B) blind intercostal nerve block (n = 24). After nerve block in both groups, pain intensity at 3, 6, 12, and 24 hours after surgery, upper limb paresthesia, frequency of nausea and vomiting, shortness of breath, hematoma, and the length of hospital stay were assessed. RESULTS: Patients who received the ultrasound-guided type-II pectoral nerve block had a greater reduction in pain intensity (24 h after surgery), opioid use (24 h after surgery), nausea, vomiting, and hospital stay than those who received ICNB, whereas the recovery time did not differ between the study groups. CONCLUSIONS: The pectoral nerve block under ultrasound guidance, compared to the intercostal nerve block, in transgender patients can reduce the required dosage of opioids within 24 hours, pain intensity within 24 hours after surgery, the incidence of postoperative nausea, and vomiting, and the hospital stay of patients.

Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development.

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like's newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like's functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.

A study to assess the quality of information in referral letters to the orthodontic department at Kingston Hospital, Surrey.

AIM: To assess the quality of information included in referral letters sent to the orthodontic department at Kingston Hospital, Surrey, UK. METHODS: Referral letters sent by both general dental practitioners (GDPs) and specialist orthodontists were analysed retrospectively in order to ascertain the percentage meeting the inclusion criteria as suggested by Mossey (2006) and the British Orthodontic Society (2008) for the quality of information included in an ideal orthodontic referral letter. Thirty-five consecutive letters sent between May and September 2005 and 206 letters sent in the same period in 2008 were collected by hand and analysed against the inclusion criteria. The numbers of referral letters received from GDPs, specialist orthodontists, and others sources were also determined. RESULTS: Most of the referrals sent in 2005 and 2008 included 40-50% of the referral inclusion points. This was despite an almost twofold increase in the number of referral letters received from specialist orthodontic practitioners in 2008. The majority of the letters, from both GDPs and specialists, did not include details of the oral hygiene or caries status of the patient, or an indication of their motivation towards treatment. None of the referral letters included a plaque score. CONCLUSION: The main weaknesses in the quality of information provided in referral letters were that in more than 80% of the letters there was no mention of the patient's medical history and no comment on caries status, the standard of oral hygiene, patient motivation for treatment, or an Index of Orthodontic Treatment Need score. The quality of information included in referral letters sent to Kingston Hospital orthodontic department needs to be improved.

Retrospective study to determine the change in referral pattern to St George's Hospital Orthodontic Department before and after the 2006 NHS Dental Contract changes.

AIM: The aim of this study was to determine the possible effects of the 2006 National Health Service General Dental Services contract changes on the referral pattern to the orthodontic department at St George's Hospital, South West London. METHOD: This study was carried out on a retrospective basis. The notes of consecutive patients referred between 1st May and 30th September in 2005 and 2008 were assessed, and the patient's Index of Orthodontic Treatment Need (IOTN) and the source of referral noted. RESULTS: The total numbers of referrals increased from 260 in 2005 to 405 in 2008. The number of referrals from general dental practitioners decreased slightly from 165 to 156, as did the numbers of referrals from other sources, such as tertiary referrals. The number of referrals made by specialist practitioners increased from 41 in 2005 to 207 in 2008, representing an increase from 16% to 51% of overall referrals. Overall, the number of patients being referred with an IOTN dental health component grade of 5 increased from 27% to 55%. CONCLUSION: The increase in referrals from specialist practitioners may be partly due to the changes brought to the commissioning of orthodontic services for specialist practitioners. Overall, the number of cases being referred with IOTN grades 4 and 5 remains high at St George's Hospital, indicating that appropriate referrals are being made.

Ankyrin repeat-containing N-Ank proteins shape cellular membranes.

Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.

Cobl-like promotes actin filament formation and dendritic branching using only a single WH2 domain.

Local actin filament formation powers the development of the signal-receiving arbor of neurons that underlies neuronal network formation. Yet, little is known about the molecules that drive these processes and may functionally connect them to the transient calcium pulses observed in restricted areas in the forming dendritic arbor. Here we demonstrate that Cordon-Bleu (Cobl)-like, an uncharacterized protein suggested to represent a very distantly related, evolutionary ancestor of the actin nucleator Cobl, despite having only a single G-actin-binding Wiskott-Aldrich syndrome protein Homology 2 (WH2) domain, massively promoted the formation of F-actin-rich membrane ruffles of COS-7 cells and of dendritic branches of neurons. Cobl-like hereby integrates WH2 domain functions with those of the F-actin-binding protein Abp1. Cobl-like-mediated dendritic branching is dependent on Abp1 as well as on Ca2+/calmodulin (CaM) signaling and CaM association. Calcium signaling leads to a promotion of complex formation with Cobl-like's cofactor Abp1. Thus, Ca2+/CaM control of actin dynamics seems to be a much more broadly used principle in cell biology than previously thought.

Assessment of mouse oocytes ultrastructure following vitrification before and after in vitro maturation / Evaluación de la ultraestructura de los ovocitos de ratón después de la vitrificación antes y después de la maduración in vitro

SUMMARY: Vitrification is a physical process in which the concentrated cryoprotectant solution after exposure to extreme cold without ice crystal formation in living cells to be converted glassing state. In this study, maturation rate and ultrastructure of mouse oocytes followed by vitrification before or after in-virto maturation (IVM) were evaluated. A total of 373 germinal vesicle oocytes were obtained from ovaries and divided into three fresh IVM, IVM vitrified, vitrified IVM groups. Ten metaphase II oocytes were obtained from uterine tubes and considered as the control group. Oocytes in vitrified groups were vitrified by Cryotop using vitrification medium and kept in liquid nitrogen. The maturation media was a-MEM supplemented with rFSH + hCG. After 24-48 h of incubation, the oocytes were investigated for nuclear maturation and ultrastructural changes using transmission electron microscopy (TEM). The oocyte maturation rate in vIVM group was significantly lower than IVMv group, when the two groups were compared with vIVM had the highest maturity. The evaluation ultrastructure of the four groups showed that the number of cortical granules, microvilli and mitochondria-SER aggregates in vIVM group were lowest and the highest amongst the number of vacuoles. Zona pellucida was darker than the control group in two freeze groups vIVM and IVMv. Most similar groups to the control group were group vIVM, Group IVMv and ultimately vIVM group, respectively. According to the results, IVM procedure is more efficient when it is performed before oocyte vitrification.

RESUMEN: La vitrificación es un proceso físico en el que la solución concentrada de crioprotectores, después de la exposición al frío extremo sin formación de cristales de hielo en las células vivas, se convierte en estado de cristal. En este estudio, se evaluaron la velocidad de maduración y la ultraestructura de los ovocitos de ratón seguidos por la vitrificación antes o después de la maduración in vitro (IVM). Se obtuvieron un total de 373 ovocitos, de vesículas germinales de ovarios, y se dividieron en tres grupos de IVM vitrificados, IVM e IVM frescos. Diez ovocitos metafase II se obtuvieron a partir de tubas uterinas y se consideraron como el grupo de control. Los ovocitos en grupos vitrificados fueron vitrificados por Cryotop usando medio de vitrificación y mantenidos en nitrógeno líquido. El medio de maduración fue a-MEM suplementado con rFSH + hCG. Después de 24-48 h de incubación, fueron observados en los ovocitos la maduración nuclear y cambios ultraestructurales utilizando microscopía electrónica de transmisión (MET). La tasa de maduración de los ovocitos en el grupo vIVM fue significativamente más baja que en el grupo IVM v, cuando los dos grupos se compararon con los que tenían la mayor madurez. La evaluación de la ultraestructura de los cuatro grupos mostró que el número de gránulos corticales, microvellosidades y acúmulos de mitocondrias-SER en el grupo vIVM fue el más bajo y el más alto entre el número de vacuolas. La zona pelúcida fue más oscura en dos grupos de congelación vIVM e IVMv, que en el grupo control. La mayoría de los grupos, similares al grupo de control, fueron los grupos vIVM, IVMv y,finalmente, el grupo vIVM, respectivamente. De acuerdo con los resultados, el procedimiento de IVM es más eficiente cuando se realiza antes de la vitrificación de ovocitos.