YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Deep-Manager: a versatile tool for optimal feature selection in live-cell imaging analysis.

One of the major problems in bioimaging, often highly underestimated, is whether features extracted for a discrimination or regression task will remain valid for a broader set of similar experiments or in the presence of unpredictable perturbations during the image acquisition process. Such an issue is even more important when it is addressed in the context of deep learning features due to the lack of a priori known relationship between the black-box descriptors (deep features) and the phenotypic properties of the biological entities under study. In this regard, the widespread use of descriptors, such as those coming from pre-trained Convolutional Neural Networks (CNNs), is hindered by the fact that they are devoid of apparent physical meaning and strongly subjected to unspecific biases, i.e., features that do not depend on the cell phenotypes, but rather on acquisition artifacts, such as brightness or texture changes, focus shifts, autofluorescence or photobleaching. The proposed Deep-Manager software platform offers the possibility to efficiently select those features having lower sensitivity to unspecific disturbances and, at the same time, a high discriminating power. Deep-Manager can be used in the context of both handcrafted and deep features. The unprecedented performances of the method are proven using five different case studies, ranging from selecting handcrafted green fluorescence protein intensity features in chemotherapy-related breast cancer cell death investigation to addressing problems related to the context of Deep Transfer Learning. Deep-Manager, freely available at https://github.com/BEEuniroma2/Deep-Manager , is suitable for use in many fields of bioimaging and is conceived to be constantly upgraded with novel image acquisition perturbations and modalities.

Observational studies on the efficacy of carbamazepine and ascorbyl palmitate in managing trigeminal neuralgia.

OBJECTIVE: Ascorbyl palmitate is a fat-soluble ester of vitamin C and is used as an antioxidant food additive. While literature reports that ascorbyl palmitate can prevent exacerbation of pain and improve the quality of life of patients suffering from pain, this is not yet supported by clinical trial data. Our study aimed to investigate the effectiveness of ascorbyl palmitate in managing trigeminal neuralgia. PATIENTS AND METHODS: This study was carried out in a single-centre clinical trial in which subjects suffering from trigeminal neuralgia (N=11) were included. All patients were on carbamazepine when first included and, after washout period, received Ascorbyl palmitate. Eligible patients had the most severe trigeminal neuralgia pain in the oral cavity or pain on touching trigger zones, aged 20 years or older, were capable of proper assessment of the severity of pain and their condition, and had experienced multiple episodes of intraoral pain for at least 3 months with a pain intensity of more than 4 points on the numerical rating scale. The Brief Pain Questionnaire was used to evaluate patient's quality of life. RESULTS: A total of 11 patients were included with a mean age 55.36±10.67 years (7 males, 4 females). Most patients had compression by the superior cerebellar artery and vascular loops upon magnetic resonance examination. The mean numerical rating scale score for carbamazepine after one month was 7.9±0.56 (95% CI 7.49, 8.30). Similarly, for ascorbyl palmitate was 5.5±1.50 (95% CI 4.42, 6.57) (p<0.001). CONCLUSIONS: Ascorbyl palmitate can be used as an adjunct intervention in managing trigeminal neuralgia pain. According to the results, ascorbyl palmitate prevents frequent exacerbation of pain and improves patient quality of life.

Open reduction and internal fixation without rigid maxillomandibular fixation: evidence based or merely a surgical dictum? A comparative pilot study on 24 cases.

OBJECTIVE: The objective of the study was to evaluate the results and immediate postoperative complications following open reduction and internal fixation of mandibular fractures with or without postoperative maxillo-mandibular fixation MATERIALS AND METHODS: The study spanned over a period of 24 months, extending from October 2015 to October 2017. The study sample comprised 24 subjects between the age range of 18 to 65 years. They were randomly divided into two groups: Group A included subjects in whom open reduction and internal fixation was followed by maxilla-mandibular fixation for 15 days, and Group B subjects in whom only open reduction and internal fixation was done, followed by immediate mobilization. The outcomes evaluated were swelling, pain, simplified oral hygiene index and occlusion. The subjects were followed for all these outcomes on 1st, 7th and 15th days. The occlusion was assessed for 5 days. Any other intra/post-operative complications were additionally noted. RESULTS: There was no statistical difference between the groups for swelling, pain and occlusion. The patients with postoperative maxilla-mandibular fixation had poorer oral hygiene when compared to the other group (p<0.001). CONCLUSIONS: The use of maxilla-mandibular fixation post open reduction and internal fixation seems to offer no additional benefits to the patients. According to the results of the study, this traditional surgical dictum seems to be used by the surgeons due to the lack of any scientific evidence. However, further studies should be conducted to confirm this statement.

Clinical effectiveness of Lactobacillus reuteri in the treatment of peri-implant diseases: a systematic review and meta-analysis.

In the last decades, the presence of peri-implant diseases (PD) has increased. One of the therapies currently used is probiotics with Lactobacillus reuteri (LR). The aim of this article is to determinate, through a systematic review and meta-analysis, the clinical effectiveness of LR in the treatment of PD. We searched the literature until January 2021, in the biomedical databases: Pubmed, Embase, Scielo, Science Direct, Scopus, SIGLE, LILACS, Google Scholar and Cochrane Central Registry of Clinical Trials. The selection criteria of the studies were: randomized controlled clinical trials, without language and time restriction, reporting the clinical effects (depth to probing, plaque index and bleeding index) of the LR in the PD treatment. The risk of study bias was analyzed through the Cochrane tool for randomized studies using Review Manager software. The search strategy resulted in 6 articles of which four investigated peri-implantitis and three peri-implant mucositis. All studies reported that there was a difference in the depth of the probing in the treatment of PD, in favor of the group using LR, though not always achieving significance. The use of LR can be clinically effective in terms of pocket depth reduction in the treatment of PD.

Role of probiotics in adjunct to non-surgical periodontal therapy in patients with chronic periodontitis: a systematic review and meta-analysis.

The objective of this study was to establish the significance of probiotic usage, both as a preventive as well as a therapeutic strategy for the management of periodontal disease. It also substantiates the existing studies of single/combined bacterial strain for exhibiting variable ecological impact on oral bacteria. Data sources included literature searches of PubMed (MEDLINE), Scopus, Embase, CENTRAL and Web of science databases for placebo controlled randomized clinical trials of SRP with orally administered probiotics in any form as an adjunct. Data extraction was conducted and information from the included studies was tabulated according to the study designs, form of drug delivery, main outcomes, and clinical parameters. Data collected were based on the focused question outlined for the present systematic review. The reviewers cross-checked all extracted data. CAL and PD were assessed as the primary outcome to compare the effectiveness of adjunctive probiotic therapy in addition to SRP. Fourteen clinical studies were included and demonstrated efficacy in reducing periodontal probing depth (PPD) and gaining clinical attachment level (CAL), between probiotics and SRP/placebo. Adjunctive probiotic therapy in addition to SRP leads to decrease in probing depth and clinical attachment gain in chronic periodontitis patients. However, further high-quality randomized clinical trials with microbiological outcomes are required to fortify the conclusion.

Delayed post-extraction implants placed using a modified Summers technique: Preliminary results of a single cohort study.

The aim of this retrospective case series was to evaluate the clinical and radiographic outcomes of the patients that underwent implant surgery with a modification of the sinus lift summers protocol. Forty healthy patients in need for oral rehabilitation with dental implants were included in this study. Inclusion criterion was the need for extraction of one compromised tooth due to persistent abscess/ periodontitis/cyst in the atrophic posterior maxilla region. The treatment consisted of two stage surgery for all patients. In the first stage, after tooth extraction, the sockets were preserved with allogenic bone graft and equine collagen membrane. After 4-5 months, 40 implants with a sandblasted surface, were inserted with osseodensification technique and a modification of the Summers sinus lift protocol for fracturing the sinus floor. The implant survival rate was the primary outcome. Intra- and postoperative complications were additional criteria for success. The mean follow-up from implant surgery was 28.0±7.3 (standard deviation) months (range 17.8-43.4 months). One implant was lost before the delivery of the prosthesis. The overall implant survival rate was 97.5%. The overall mean peri-implant marginal bone level change after 6 and 12 months of function was, respectively, 0.26±0.24 mm (95% CI: 0.19, 0.34 mm) and 0.71±0.36 mm (95% CI: 0.60, 0.82 mm). Marginal bone loss was statistically significant at both time frames respect to implant placement, and also the difference between 6 and 12 months was significant (p<0.001 in both cases). No biological nor mechanical complications were recorded throughout the observation period. As a conclusion, the technique presented in this cohort study can be an effective and safe alternative to standard maxillary sinus floor augmentation procedures and immediate implant insertion protocol, especially in cases of periodontitis and infected sites, which can represent a high risk for implant failure in patients with atrophic posterior maxilla.

Contribution of Indian researchers in high impact periodontal journals: a bibliometric analysis of last three years.

Bibliometric Analysis researches and analyses the quantitative data derived from scientific publications through the empirical evidence of scientific activity generated by collaborating authors through the final product of their research: the scientific article. In scientific society, the concept of impact factor is probably the most widely used in bibliometric construction. To assess the scientometrics of three high-impact factor periodontal journals and identify the contribution of India in these most productive journals over three years (Jan 2018 - Dec 2020) and to know the most influential topics researched. A retrospective observational study was conducted for the Journal of Clinical Periodontology, Journal of Periodontology, and Journal of Periodontal Research. All issues of 2018, 2019, and 2020 were electronically and hand searched for the following parameters: Number of papers, affiliated organizations, and countries, topics reported, and contribution of Indian authors. The data were organized and analyzed with descriptive statistics using SPSS software (version 21.0). In total 469 articles were published by Journal of Periodontology, followed by 454 articles in Journal of Clinical Periodontology and 287 articles in Journal of Periodontal Research. In all the three journals, China had the maximum contributions, succeeded by USA. India has published maximum number of articles in the Journal of Periodontal Research. When analysed, although less as compared to the western counterparts, an increasing trend in the publications is seen in case of India.

Evaluation of mental foramen and inferior alveolar nerve canal and its relationship to adjacent anatomical landmarks using cone-beam computer tomography.

The objective of the research was to evaluate the location, size, variability, and morphologic features of mental foramen (MF) and the inferior alveolar nerve canal (IAN) on cone-beam CT. We evaluated the morphologic findings of mental foramen (MF) and inferior alveolar nerve (IAN) canal of 88 mandibular hemiarches of 65 Caucasian subjects (35 males, 30 females; age range 25-75 years) using cone beam CT. The most common horizontal position of MF was type 3 (53.4%), followed by type 4 (39.8%), type 1 (2.3%), type 2 (2.3%), and type 5 (2.3%). Regarding the vertical position, in 71.6% of cases (63/88) we found type 3 position, followed by type 2 (22.7%) and type 1 (5.7%). MF presented as oval in 51.1% and round in 42%, with double oval and triple foramens having been observed in 5.7% and 1.1% respectively. In 36.9% of cases, we found an anterior loop of the IAN. The mean depth of MF was 6.12±1.65mm; width and height were 3.7±0.83mm and 3.14±0.78mm. Width and height of the IAN distal to MF were 2.27±0.53mm and 2.74±0.51mm, while those of the incisive nerve canal mesial to MF were 1.37±0.44mm and 1.54±0.58mm, respectively. An increase in the width of MF was correlated to oval shape (r=0.45; P < 0.01), and there was a low but significant correlation (r=0.23; P < 0.05) between the round shape of MF and the size of the IAN. MF shape appears to be correlated to MF width and size of the IAN. The individual anatomical variability of this structure is a factor that must be considered when dealing with mandibular surgery.

Effect of concentrated growth factors on quality of life of patients undergoing implant therapy: a cohort study.

Pain, bad taste, and impaired daily activity after implant therapy are common sequelae. Concentrated growth factors (CGF) are a platelet concentrate with a favourable effect on wound healing, but there is still no evidence regarding its potential benefits for reducing postoperative pain and symptoms. Therefore, aim of this prospective comparative study was to determine the effect of CGF on quality of life (QoL) of patients after implant therapy. Fifty-two consecutive patients with one missing mandibular molar were included in the study and alternatively assigned to two groups. Control group received standard implant treatment, and test group received CGF associated with implants. Standard periapical radiographs were taken before and after procedure. Post-operative care consisted of 0.2% chlorhexidine digluconate solution twice daily for 10 days. A QoL questionnaire (OHIP-14) for bad taste, pain and limitation in daily activities was filled and returned one week post-operatively. Daily pain was also assessed through Visual Analogue Scale (VAS) on a 1-100 scale. Parametric test (chi-square) was performed to compare the results of the questionnaire between the two groups using STATA statistical software. All patients correctly filled and returned the questionnaire. Significantly higher proportions of patients of test group reported no bad taste, pain, and limited activity, (24/26, 13/26, and 25/26, respectively) respect to control. Postoperative pain with VAS score was significantly lower in the test group on day 1, 2, and 3 as compared to control. CGF positively influenced QoL when associated with implant rehabilitation of mandibular molars, minimizing post-operative discomfort.

Discovering the hidden messages within cell trajectories using a deep learning approach for in vitro evaluation of cancer drug treatments.

We describe a novel method to achieve a universal, massive, and fully automated analysis of cell motility behaviours, starting from time-lapse microscopy images. The approach was inspired by the recent successes in application of machine learning for style recognition in paintings and artistic style transfer. The originality of the method relies i) on the generation of atlas from the collection of single-cell trajectories in order to visually encode the multiple descriptors of cell motility, and ii) on the application of pre-trained Deep Learning Convolutional Neural Network architecture in order to extract relevant features to be used for classification tasks from this visual atlas. Validation tests were conducted on two different cell motility scenarios: 1) a 3D biomimetic gels of immune cells, co-cultured with breast cancer cells in organ-on-chip devices, upon treatment with an immunotherapy drug; 2) Petri dishes of clustered prostate cancer cells, upon treatment with a chemotherapy drug. For each scenario, single-cell trajectories are very accurately classified according to the presence or not of the drugs. This original approach demonstrates the existence of universal features in cell motility (a so called "motility style") which are identified by the DL approach in the rationale of discovering the unknown message in cell trajectories.

The influence of spatial and temporal resolutions on the analysis of cell-cell interaction: a systematic study for time-lapse microscopy applications.

Cell-cell interactions are an observable manifestation of underlying complex biological processes occurring in response to diversified biochemical stimuli. Recent experiments with microfluidic devices and live cell imaging show that it is possible to characterize cell kinematics via computerized algorithms and unravel the effects of targeted therapies. We study the influence of spatial and temporal resolutions of time-lapse videos on motility and interaction descriptors with computational models that mimic the interaction dynamics among cells. We show that the experimental set-up of time-lapse microscopy has a direct impact on the cell tracking algorithm and on the derived numerical descriptors. We also show that, when comparing kinematic descriptors in two diverse experimental conditions, too low resolutions may alter the descriptors' discriminative power, and so the statistical significance of the difference between the two compared distributions. The conclusions derived from the computational models were experimentally confirmed by a series of video-microscopy acquisitions of co-cultures of unlabelled human cancer and immune cells embedded in 3D collagen gels within microfluidic devices. We argue that the experimental protocol of acquisition should be adapted to the specific kind of analysis involved and to the chosen descriptors in order to derive reliable conclusions and avoid biasing the interpretation of results.

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells.

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Calcium signals and FGF-2 induced neurite growth in cultured parasympathetic neurons: spatial localization and mechanisms of activation.

The growth of neuritic processes in developing neurons is tightly controlled by a wide set of extracellular cues that act by initiating downstream signaling cascades, where calcium signals play a major role. Here we analyze the calcium dependence of the neurite growth promoted by basic fibroblast growth factor (bFGF or FGF-2) in chick embryonic ciliary ganglion neurons, taking advantage of dissociated, organotypic, and compartmentalized cultures. We report that signals at both the growth cone and the soma are involved in the promotion of neurite growth by the factor. Blocking calcium influx through L- and N-type voltage-dependent calcium channels and transient receptor potential canonical (TRPC) channels reduces, while release from intracellular stores does not significantly affect, the growth of neuritic processes. Simultaneous recordings of calcium signals elicited by FGF-2 at the soma and at the growth cone show that the factor activates different patterns of responses in the two compartments: steady and sustained responses at the former, oscillations at the latter. At the soma, both voltage-dependent channel and TRPC blockers strongly affect steady-state levels. At the growth cone, the changes in the oscillatory pattern are more complex; therefore, we used a tool based on wavelet analysis to obtain a quantitative evaluation of the effects of the two classes of blockers. We report that the oscillatory behavior at the growth cone is dramatically affected by all the blockers, pointing to a role for calcium influx through the two classes of channels in the generation of signals at the leading edge of the elongating neurites.

Spatiotemporal regulation of the Pak1 kinase.

Pak1 (p21-activated kinase 1) is a key regulator of the actin cytoskeleton, adhesion and cell motility. Such biological roles require a tight spatial and kinetic control of its localization and activity. We summarize here the current knowledge on Pak1 dynamics in vivo. Inactive dimeric Pak1 is mainly cytosolic. Localized interaction with the activators Cdc42-GTP and Rac1-GTP stimulates the kinase at the sites of cellular protrusions. Moreover, Pak1 is dynamically engaged into multiprotein complexes forming adhesions to the extracellular matrix. Cutting edge microscopy technologies on living cells are finally shedding light on the intricate spatiotemporal mechanisms regulating Pak1.

Sonographic evaluation of subacromial space.

GOAL OF THE STUDY: The purpose of this study is to compare the accuracy of sonographic to radiographic measurements of subacromial space, and verify its variations in relation to acromial morphology, age, sex and rotator cuff pathologies. MATERIALS AND METHODS: As a result, we have compared a radiographic examination to sonographic examination, each measuring the subacromial space in 200 random shoulders, with a personal method. The sonographic examination was performed by using a HDI 5000 ultrasound scanner Sono-CT with 7.5 MHz linear array transducer. No stand-off pad was utilized. RESULTS: The statistical analysis of the data derived from the two measurements was not sufficient to conclude that the two techniques are different (p>0.8). They also correspond with the radiographic morphology of the acromion. The size of subacromial space was related to the acromial morphology, female gender, and rotator cuff pathology, however, it was not related to age. DISCUSSION AND CONCLUSIONS: Our results clearly show that sonographic measurements are very close to those obtained by X-ray (p>0.8). The Bland-Altman analysis showed that for all groups, the were small enough to give us confidence that the sonographic technique may be used in place of the radiographic one for clinical purposes. One-way ANOVA showed that sonographic measurements were statistically different among the four groups (p<0.05). The sonography demonstrated precision, accuracy and carefulness in the measurement of the subacromial space.

Synovial hemangioma. Description of a case.

Intra-articular angioma, or vascular hamartoma, is a tumor-like lesion essentially made up of anomalous blood vessels localized in the joint capsule, in the synovial membrane, or in both structures. There are two anatomical varieties: one circumscribed localized and one extended. There seems to be predilection for the female sex, and age of onset ranges from birth to 20-30 years of age; it occurs more frequently in the knee, less in the elbow, in the wrist and in the ankle. Symptoms, which often begin after local trauma, include joint swelling and pain, both characterized by discontinuity and long duration. Sometimes there is an increase in swelling, repeated episodes of hemarthrosis (or hydrarthrosis), forced position and functional limitation of the joint, increase in skin temperature. Final diagnosis can only be obtained with histological examination, although MRI may be useful and arteriography clearly reveals it, if the harmatoma is extended enough and communicating with the circulation. Histological examination carried out on fragments of tissue taken in loco reveals a labyrinthine agglomerate of fissures and lengthening cavities, with walls that are generally thick and of a venous-like anomalous type. The course of the disease is slow, with periods of remission of local symptoms of varying duration. Treatment is surgical and it consists in complete removal when possible of the angiomas. The results, particularly in the localized form, are satisfactory; recurrence is infrequent and it is generally due to incomplete excision of the neoformation.

Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch.

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability.

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.

Engineering temperature-sensitive SH3 domains.

BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks. SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences. RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive. Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature. Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins. Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells. CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo. Such mutants will be valuable in dissecting SH3-mediated signaling pathways. Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions.