It is known that high-intensity exercise can cause inflammation and damage in muscle tissue, and in recent years, physical therapists and fitness professionals have begun to use foam rolling as a recovery method to improve performance. Despite the lack of basic science studies to support or refute the efficacy of foam rolling, the technique is very widely used in the sports world. In this respect, we investigated whether foam rolling could attenuate muscle damage and inflammation. Female Wistar rats were assigned to control (C), foam rolling (FR), notexin without foam rolling (N) and notexin with foam rolling (NFR) groups. A 4.5 x 2 cm foam roller was used to massage their hind legs (two 60-second repetitions twice a day for 3 days). Motor function tests  were used. We detected an increase in time and foot faults when crossing a beam in the N group compared to C and FR rats. In contrast, a significant decrease was detected in both tests in NFR compared to N rats. Muscle power was measured with a grip strength test and better performance was detected in NFR rats compared to N rats. Furthermore, an increase of pro-inflammatory proteins was noted in the N group, while there was a decrease in the NFR group. On the contrary, an increase in PPAR-γ (anti-inflammatory protein) in the NFR group compared to the N group demonstrates the anti-inflammatory properties of the foam rolling technique. In summary, applying foam rolling after damage has benefits such as an increase in anti-inflammatory proteins and a reduction of pro-inflammatory proteins, resulting in muscle recovery and better performance. It is known that high-intensity exercise can cause inflammation and damage in muscle tissue, altering immune functionIn recent years, physical therapists and fitness professionals have begun to use foam rolling as a recovery method to improve performance. Foam rolling is a commonly used technique that requires individuals to use their own body mass on a foam roller to apply pressure to the soft tissueDifferent studies in which foam rolling has been used after performing physical tests or applying a delayed-onset muscle soreness induction protocol have shown that the use of this instrument makes it possible to reduce perceived fatigue and muscle painDespite this, there is still little evidence regarding the influence of foam rolling at a physiological level that can explain the aforementioned effects. Specifically, some authors have observed that after applying the technique there was a decrease in pulse wave velocity and an increase in the concentration of nitric oxide, concluding that it could provide benefits in terms of arterial function2 neurotoxin isolated from snake venom, has been described as causing complete destruction and rapid regeneration of muscle fibers and providing an effective and reliable model of skeletal muscle injury/regeneration in the mouse2+ overload and activation of Ca2+-dependent proteasesDespite the lack of basic science studies to support or refute the efficacy of foam rolling, the technique is very widely used in the sports world. In this respect, the present study aims to further explore the effects of foam rolling on muscle damage and inflammation. An experimental design was carried out applying foam roller technique during 3 days after Notexin-induced injury. Notexin is a presynaptic phospholipase AThis study tested the hypothesis that the application of foam rolling after notexin-induced muscle damage could act as a protective technique, so we studied its effects on rat muscle, looking at inflammation, changes in cell death and changes in exercise performance with the samples obtained after 6 training sessions.Twenty female Wistar rats  were randomly divided into 4 groups with 5 in each group, including: control (C), foam rolling (FR), notexin without foam rolling (N) and notexin with foam rolling (NFR). When notexin was used, only one leg was injected.All the rats were fed ad libitum on a standard diet and were kept on a 12-h light/12-h dark cycle, keeping the room temperature at 22ºC. The rats were numb before the injection with notexin, the blood draw and the application of a foam roller with inhalation anesthesia (5% inofluoran by induction and 2% maintenance). At the end of the study, the animals were euthanized  for removal of the quadriceps muscle. Death certification was due to heartbeat and stiffness. For the experiments, the estral phase chosen was diestro. It was identified by vaginal lavage and the appearance of rounded cells of small size and with very few keratinized cells.All animal procedures were carried out in accordance with the European legislation on the use and care of laboratory animals (EEC 86/609). Experimental research on rats was performed with the approval of the ethics committee on animal research of the University of Valencia, Spain (Code: 2018/VSC/PEA/0004).A high-density foam roller was applied to the hind legs of 10 rats (FR and NFR groups). The massage was applied by the veterinarian in two 60-second repetitions, twice a day (at 10 a.m. and 5 p.m.) for 3 days. The pressure applied was 25% of the rat's weight. To know the pressure exerted, rats were weighted before and during the application of foam roller. The measures of the foam roller were 4.5 x 2 cm.To induce muscle injury, 200 µl of notexin was injected intramuscularly at 10 µg/ml in the quadriceps, only in one leg of the rat. Notexin was injected 24 h before foam roller application. The control rats were injected with 200 µl of saline solution.Plasma was obtained from the rats and used to determine IL-1β and TNF-α concentration (pg/ml) using ELISA kits  . The blood was obtained from the lateral vein of the tail (1 ml) prior anaesthesia. Plasma was obtained and for each test 100 µl was used.The protein extracts from the quadriceps were mixed with equal volumes of SDS buffer  2-mercaptoethanol, 1% bromophenol blue and 19% glycerol) and then boiled for 5 min. The protein concentration was determined using a modified Lowry methodThe tubulin protein was used as control. Loading controls are essential for proper interpretation of western blots and can be used to normalize the levels of protein detected by confirming that protein loading is the same across the gel. All other reagents are of analytical or culture grade purity.Motor function was determined by two different tests: Balance Beam Test and Grip Strength; in the first test, the rats' ability to pass along a narrow beam to reach a dark box was evaluated to assess the motor coordination of animals. To force the rats to pass along the beam, a white light illuminated the beginning of the beam. The wooden beam (1 x 150 cm) was elevated to a height of 1.5 m above the floor. The time required crossing to the escape box at the other end of beam and the number of forelimb and hind-limb paw slips were recorded. A paw slip was defined as any paw coming off the top of the beam or any limb use on the side of the beam. On the day of the test, four trials were performed before recording the results. The goal was to familiarize the rats with the beam and to train them in the presence of the dark box at the end of the beam. The narrow beam and the dark box were cleaned after each trial with ethanol.In a second type of test, grip strength was measured non-invasively by taking advantage of the rat's instinctive tendency to grab as they are gently pulled backward. This method can be used to test for limb grip using a horizontal bar with a small diameter (0.1 cm), which is attached to a force transducer. The length of time the rat was able to hold the bar was recorded. This latency to grip loss is considered as an indirect measure of grip strengthThe rat was allowed to hold, with the forepaws, a steel wire (2 mm in diameter and 80 cm in length), placed at a height of 50 cm over a cushion support. The length of time the rat was able to hold the wire was recorded. This latency to grip loss is considered as an indirect measure of grip strengthStatistical analyses were performed using the Statistical Package for the Social Sciences . The data was analysed by parametric ANOVA (there is homogeneity of the variances). In addition to this, Scheffe method was used to determine post-hoc pair-wise comparisons. A p-value less than 0.05 were considered to indicate statistical significance.The rats' ability to pass along a narrow beam showed using the beam walking test. C and FR did not display any general motor and muscular impairment, as demonstrated by unaffected grip strength and toe hold ability when crossing a beam in the beam walking test Figure . In the Alterations in motor coordination may be related to inflammation and oxidative stress in N rats. Thus, we subsequently determined apoptosis protein expression by western blot. Our results showed that expression of Cytochrome c was significantly higher in N rats than in C rats (p<0.05) Figure a. Also, IL-1β and TNF-α pro-inflammatory proteins. Serum from the rats  was assayed to determine the cytokine IL-1β and the mediator TNF-α by ELISA. Figure We noted a significantly increase in NF-κB expression in the N group compared with the C and foam rolling groups (p<0.05) Figure a. The NFAnti-inflammatory proteins, such as PPAR-γ (peroxisome proliferator-activated receptor γ), down-regulate gene expression of pro-inflammatory proteins. Figure An increase in p38 and p-p38 expression is related to an increase in cell differentiation and release of cytokines for cell-to-cell communication. Here, a significantly increase in both proteins was detected in the NFR group compared to C and FR rats (p<0.05) Figure a and 6b,Vascular endothelial growth factor (VEGF) protein expression was assayed to determine vascular endothelial changes in rat quadriceps. No changes in VEGF protein expression were detected in the C, FR and N groups. However, a significantly increase in VEGF expression by applying foam rolling after the notexin injection demonstrated a rise in endothelial growth factors, producing an improvement in the vascular muscle (p<0.05) Figure .The present study aims to demonstrate the physiological changes produced by the use of foam rolling. To our knowledge, this is the first study that makes it possible to justify the biomolecular effects on recovery and performance after applying this technique. Under the hypothesis that foam rolling could act as a protective technique, we studied its effects on rat muscle, looking at inflammation and changes in cell death.So far, the application of foam rolling in humans has been shown not to have a negative effect on performanceWe noted a destruction of the cell nucleus, apoptosis and necrosis compared with untreated cells. The signalling of p38 is necessary to protect cells against apoptosis and our results demonstrated a reduction in that kinase after the addition of notexin, with a significant increase after foam roller use, thus showing muscle protection after foam roller use. However, in the present study the muscle damage caused by physical exercise has not been assessed, therefore we cannot compare the damage induced by notexin with that induced by physical exercise.The exact mechanisms by which foam rolling leads to recovery from muscle damage remain to be determined, but under our conditions the foam roller caused a decrease in cell death due to apoptosis. The process of apoptosis is associated with the release of caspase-dependent proteins, such as Cytochrome c and Smac/Diablo, and AIFSmac/Diablo produces apoptosis by neutralizing one or more members of the IAP (inhibitor of apoptosis proteins) familyIn normal conditions, there is a balance of pro- and anti-inflammatory mediators in order to maintain equilibriumMoresi et al. have published that in a muscle cell culture TNF-α directly decreases total muscle protein, including adult fast-type myosin heavy chainIn situations of inflammation, such as notexin-induced muscle damage, NF-ᴋB protein expression is up-regulated. Pro-inflammatory transcription factor nuclear factor κB (NF-κB) is formed by different subunits and located in the cell cytoplasm. In non-activated conditions it is bound to its inhibitor IκB. The release of NF-κB, after activation by pro-inflammatory mediators produces translocation to the nucleus and subsequent transcription of pro-inflammatory proteinsInducible cyclooxygenase (COX-2) and the involvement of neurotrophic factors have been related to delayed-onset muscle sorenessIt has been shown in the literature that notexin produces inactivation of the MAP kinase cascadeBecause of the possible protective mechanism against inflammation after foam rolling, a decrease in pro-inflammatory and an increase in anti-inflammatory proteins may be activated by foam rolling. Indeed, PPAR-γ plays a fundamental role in the immune response because it decreases the expression of pro-inflammatory genesOn the other hand, vascular endothelial growth factor (VEGF) is a paracrine factor that plays a major role in the promotion of angiogenesis, improving cellular survival, inducing proliferation and increasing the migration and invasion of endothelial cells. VEGF is also induced by PPAR-γ in cardiac myofibroblastsIn summary, our study demonstrates an increase in apoptosis and inflammation after notexin injection Figure a and conThis study demonstrates that foam rolling prevents the increase in Cytochrome c, AIF and Smac/Diablo expression induced by notexin in quadriceps muscles, indicating that foam rolling prevents notexin-induced cell death by apoptosis. Furthermore, a reduction in pro-inflammatory mediators  and increase in anti-inflammatory mediators (PPAR-γ) can result in recovery from notexin-induced damage. Moreover, an increase in VEGF was detected. These results would justify the use of foam rolling as a method for muscle recovery after toxic damage.


Genital tuberculosis (GTB) is an important cause of female infertility, especially in developing countries. The positive results of polymerase chain reaction (PCR) in endometrial GTB in the absence of tubal damage raise the possibility of the detection of sub-clinical or latent disease, with doubtful benefits of treatment.This study aims to evaluate the Mycobacterium tuberculosis (MTB) and Non-tubercular Mycobacterium (NTM) infection by using Real-PCR technique in the menstrual blood samples of 120 unexplained infertile women.In this cross-sectional study, 120 infertile women with unexplained infertility aged 20-35 yr old and normal hysterosalpingography findings were taken. Menstrual blood in the first 12 hr of menstruation containing the endometrial tissues from each participant was tested for MTB and NTM by Real-Time PCR. Among the selected 120 patients, only two were found to be positive for MTB infection. All remaining participants were negative for MTB infection. All participants were negative for NTM infection at the endometrium.Although, studies have indicated that PCR is a useful method in diagnosing early GTB disease in infertile women with no demonstrable evidence of tubal or endometrial involvement, our study showed that GTB is not the major problem in women with unexplained infertility. Female infertility is the outcome of a number of factors that include: problems in ovulation, tubal blockage, uterine problem, endomeriosis, pelvic disorders, endocrine disorder as well as ‘unexplained fertility’ for which medical examinations can not reveal any specific cause . UnexplaMycobacterium tuberculosis (MTB), the causative agent of genital tuberculosis involves mainly fallopian tube, endometrium and to lesser extents in ovary, cervix . Since iRapid nucleic acid amplification techniques, such as PCR has many advantages, such as only 10 CFU/ml is detectable and very small time is needed for obtaining results. Advanced PCR techniques like Real-time PCR (RT-PCR) markedly reduce the incidence of false positive results as amplification and detection takes place in the sample reaction tube . FurtherIn this present study an initiative has been taken to detect MTB and NTM by RT-PCR in the early menstrual blood and endometrial contents within it of 120 unexplained infertile females to find association of GTB with their infertility. The detection employed nested PCR for the detection of MTB and NTM. The first amplification was subjected for sequence-specific amplification of MTB and NTM and the second amplification was a nested PCR reaction in order to achieve the maximum sensitivity and specificity of the test.This cross-sectional study included the infertile women aged between 20-35 yr, who came to us for diagnosis of their GTB infection . None of the selected women had any history of endocrine disorders, polycystic ovarian syndrome or any known cause of infertility. Any women with a prior history of tuberculosis, contact history of tuberculosis and chest radiological findings were excluded from this study. Specimen collection Specimens were collected within first 12 hr of the onset of menstruation. The patients were given a sterile container, where they themselves collected the menstrual secretions, which contain a heterogeneous mixture of endometrial tissues and menstrual blood. The patients were advised not to have any physical intercourse before 48 hr of sample collection and also advised not to use any gel or cream at vaginal area prior collection of sample.DNA extractionThe Mycobacterial DNA extraction was done using commercially available kit from 3B Black Biotech Ltd. in the following steps:Sample lysisSample neutralizationAdjusting DNA binding conditionsBinding of DNAWashing silica membraneEluting highly pure DNAoC) for 15 min. After this step 100 μl neutralization buffer was added to the samples, mixed well and centrifuged for 2 min at 10,000 rpm. After centrifugation 200 μl of supernatant was transferred into a new 1.5 ml microcentrifuge tube and 250 μl of buffer was added to this for facilitating the DNA binding condition. After this step 250 μl of ethanol (96-100%) was added to samples and the mixture was vortexed vigorously (for 10-20 sec). The samples were centrifuged and endometrial tissues, present in menstrual secretion were finely chopped with sterile surgical blade. Following this 100 μl lysis buffer was added to the pellet and the sample was incubated at a boiling water bath  placed into a 2 ml collection tube. Then the spin column was centrifuged for 1 min at 10,000 rpm and the flow-through was discarded. Now the DNA has bound to the membrane. Now it needs to be washed couple of times with wash buffers and finally DNA was eluted with 50 μl elution buffer after incubating the membrane for 1 min at 70RT- PCRThe RT-PCR was done using the commercially available kit from 3B Black Biotech Ltd. This RT-PCR kit is approved by CE and IVD. The PCR Mix was prepared as per RT-PCR machine program set upWe have used Rotor Gene Q (6 plex) RT-PCR machine and the machine was programmed as per Channel selectionThe settings for channel selection were defined in the PCR machine as per Ethical considerationThe whole study protocol was approved by the Institutional Ethics committee. Oral Consent was taken from each of the participants for their clinical data can be used for research purpose.We were very strictly followed the results and data of the RT-PCR runs. If there was proper amplification of Internal Control (IC) in the TEXAS RED channel, then only it was taken for further analysis for detecting the presence of MTB or NTM. Out of our analyzed 120 cases, we have got M. tuberculosis in 2 samples. 118 were free from any kind of MTB or NTM infection. The The incidence of female infertility is rising throughout the world of which 30% of the infertile couple diagnosed with unexplained infertility , 15. AltIn this present study, we have analyzed first 12 hr menstrual blood which is reported to be a good source of endometrial tissue  from 120For example, in a cross-sectional study in Iran endometrial biopsy samples of 144 infertile women with unexplained infertility aged 20-35 yr, the PCR results of endometrial specimens were negative in all cases indicating that there was no GTB infection in these patients (Although MTB is quite prevalent in developing countries like India, but GTB is not that much common as compared to pulmonary tuberculosis . Anyway,


Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication. Centrioles are microtubule-based, radially symmetric, cylindrical structures. A pair of centrioles, and surrounding pericentriolar material, comprise the centrosome . In inteIn dividing cells, centriole generation occurs in S phase of the cell cycle when exactly two new (daughter) centrioles assemble, each next to an existing (mother) centriole  Fırat-K. The resMulticiliated cells (MCCs) of the airway, ependymal, middle ear and oviduct epithelia break the rules that govern centriole formation in dividing cells, as they assemble, depending on cell type, dozens to hundreds of centrioles in a single postmitotic cytoplasm to act as basal bodies to motile cilia  Meunier. MCC fatMechanisms that control centriole number in dividing cells consist of both centriole-intrinsic and cytoplasmic events. Limiting centriole duplication to S phase is ultimately under the control of the cell cycle machinery. Timely entry and progression through S phase is regulated by Cyclin-dependent kinase 2 (Cdk2) complexed with cyclins E or A2 . DistincInterestingly, recent insights into MCC differentiation revealed that multiple cell cycle regulators also play important roles in motile ciliogenesis . The iniTo investigate the extent to which the G1 to S phase cell cycle machinery is involved in motile ciliogenesis, we tested the role of Cdk2 using primary mouse tracheal epithelial cell (MTEC) culture, which recapitulates basal (stem) cell proliferation and subsequent MCC differentiation . Here, wWe used the MTEC culture system to test the requirement for Cdk activity during MCC differentiation . MTECs aTo test the requirement for Cdk activity, we treated differentiating MTECs with well-characterized, dose-dependent small molecule inhibitors that predominantly act on Cdk1 and Cdk2 (Cdkis). We verified the previously established ability of these Cdkis to produce cell cycle arrest in 293T/17 cells before using in MTECs (not shown). MTECs were treated from ALI + 0 to ALI + 4 d (chronic treatment) with the Cdkis Purvalanol A, Roscovitine, NU6140 and Cdk2 Inhibitor III, then labeled at ALI + 4 d with anti-acetylated α-Tubulin (ac. α-Tub) antibody to mark cilia. While untreated cultures contained many MCCs, we found that all four Cdkis blocked ciliogenesis . To charCiliogenesis arrest was fully reversible for all Cdkis, as MTECs released from ALI + 0 to +4 d Cdki treatment were robustly ciliated by ALI + 8 d . These rThe Cdkis used are known to have dose-dependent activity on multiple Cdk-cyclin complexes, and at higher doses they can also inhibit unrelated kinases . We founUpon airway epithelialization, MCC and secretory cell fates are acquired in a Notch signaling-dependent mechanism. Precursor cells in which Notch signaling is activated are diverted to the secretory cell fate, whereas cells in which Notch is not activated continue toward MCC differentiation  Figure 1TRRAP, a component of multiple histone acetyltransferase complexes was recently shown to be required for the initiation of the MCC gene expression program ‘downstream’ of the Notch signaling event (downstream in the same sense as described for Cdk2 above) . TRRAP nNext, we more closely investigated the relationship between Cdk2 and the MCC transcriptional regulators Mcidas, Myb and Foxj1. While the precise hierarchy remains unclear, Mcidas (as part of the EMD complex) is known act upstream of the other two, but there is also evidence for feedback regulation and overlap in targets . As MCC Our results place the Cdk2 requirement downstream of the Notch-dependent cell fate decision and upstream of EMD in the MCC gene expression pathway for initiating ciliogenesis. The clear temporal separation of the proliferation (preALI) and differentiation (beginning at ALI + 0 d) phases of the MTEC culture system already made it unlikely that the ciliogenesis block by Cdki treatment starting at ALI + 0 d stems from the disruption of a proliferative event. The fact that Cdk2 acts downstream of the Notch signaling event  indicateMyb is expressed first during motile ciliogenesis; then it turns on Foxj1  and is then downregulated while Foxj1 stays on  declined upon Cdki treatment, which suggests that continued Cdk2 activity is required to maintain MCC gene expression  MCCs . Althoug07) MCCs  by Weste07) MCCs . The pre07) MCCs . As we w07) MCCs , and tha07) MCCs . Thus, bCcna1, Ccna2, Ccne1 and Ccne2 during motile ciliogenesis , and this pattern can be used to identify transcripts important for the process , FOXJ1 and MYB  and acute (ALI + 3 to+4 d) Cdki treatment  downstream of cyclin binding , and it Ccna1 mutant mice. Although technical limitations  prevent us from demonstrating direct association in ciliating cells, their colocalization during ciliogenesis, similar loss of function phenotypes and the existence of Cdk2-Ccna1 complexes in other tissues . Cdks, including Cdk2, have been reported at the centrosome in dividing cells, so this is likely a localization pattern shared with MCCs. Centrosomal Cdk2 targets include Ccp110, which dissociates from the mother centriole distal end upon phosphorylation to allow primary cilium growth . Ccp110 Cdk2 knockout mouse  mice were obtained from JAX. Ccna1     , Ccne1; dge, MA)  and Foxjill, NC)  mice havill, NC) . In shorD145N-HA  cells were used for lentiviral preparation and the Luciferase and endogenous gene activation assays. mIMCD3  cells were used to test Ccna1 antibody specificity. Cells were purchased from ATCC and were assumed to be authenticated by the supplier. Cells were not specifically monitored for mycoplasma contamination, but routine DAPI staining would have revealed the presence of contamination.293T/17 . Lung tissues were fixed in 4% paraformaldehyde overnight at 4°C, rinsed in PBS, incubated in 30% sucrose, embedded in OCT compound and frozen. 10 μm cryosections were labeled as above. Samples were imaged with Leica LAS X software on a Leica SP5 or SP8 confocal microscope (Leica). For antibodies and fixation conditions, see Supplementary Table 1 in MTEC lysates were generated in triplicate from MTECs cultured in 12- or 6-well Transwells to the desired stage and harvested by scraping the Transwell surface in 1x Laemmli sample buffer. Approximately 10,000 cells per lane were loaded for SDS-PAGE, then transferred to nitrocellulose membrane. For the MTEC timecourses equal loading was verified by Ponceau S staining of the membrane. Acetylated α-Tubulin antibody labeling shows increasing signal with increased ciliogenesis as MTECs mature and thus should not be interpreted as a loading control. Mouse testis lysate was prepared using published methods  to serveFoxj1-EGFP MTECs grown on 12 6-well Transwells as previously described  in a StepOnePlus Real-Time PCR System (Thermo Fisher), and gene expression was evaluated using the ΔΔCt method. For primer sequences, see Supplementary Table 2 in cDNA was prepared from MTECs cultured on 12- or 6-well Transwells at various timepoints and with various treatments as indicated, and from MCCs (EGFP+) and non-MCCs (EGFP-) obtained by FACS from escribed . Early, The ratio of lentivirally expressed Cdk2-HA nuclear to cytoplasmic signal intensity in MCCs  was quantitated on individual image slices using ImageJ (NIH) by measuring signal intensity for manually outlined nuclear and cytoplasmic areas  and normalizing to the total measured area.Human FOXJ1 and CCNA1 promoter genomic DNA fragments see  were cloAdult mouse airway tissues were fixed in 2% glutaraldehyde, 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4  at 4°C overnight, osmicated, dehydrated and dried with a Tousimis Autosamdri-815 critical point dryer. Samples were then mounted luminal side up, sputter coated with 100 Å layer of Au/Pd and analyzed with a Hitachi S-3400N VP-SEM microscope (Hitachi) operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.Sample-size estimation: No explicit power analysis was used during the design of the study. Biological and experimental replicates  was used to generate graphs and perform statistical analyses. Pairwise comparisons were made with a two-tailed Student’s t test or an ANOVA test. For multiple comparisons, a follow up test (Dunnett’s or Bonferroni’s) was applied to correct for multiple hypothesis testing. For all cases, a p value less than 0.05 was considered significant. Error bars on graphs represent standard error. See Supplementary Table 3 in Group allocation: samples generated in an identical manner were allocated into groups based on treatment  or genotype.Source data: not applicable In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Steven Brody (Reviewer #3).Thank you for submitting your article "Cyclin-dependent kinase control of motile ciliogenesis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary-/-Ccna1 mice.The manuscript submitted by Vladar and colleagues reports a novel role for the canonical mitotic regulator Cdk2 in the control of ciliogenesis in murine airway culture systems. They claim that Cdk2 is required to initiate and maintain the MCC gene expression program during ciliogenesis. They claim that Cdk2 is coupled with cyclin A1 to initiate centriole replication in MCCs. The authors begin by assessing the effects of pharmacologic Cdk inhibitors on differentiating MTEC ALI cultures, observing that inhibition of Cdk2 produces a reversible block in the initiation ciliogenesis revealed by the absence of MCC markers by immunofluorescence imaging. The specificity of this result was established with a dominant-negative allele of Cdk2, which phenocopies pharmacologic inhibition. Subsequent data suggests ongoing Cdk2 activity (after initiation of ciliogenesis) is also required for MCCs to complete maturation into "Stage IV" or Foxj1+/Myb- cells. In order to establish epistasis relationships, MTEC ALI were treated simultaneously with Cdk2 inhibitors and the Notch antagonist DAPT or with Cdk2 inhibitors in the setting of enforced Mcidas expression. The claim is that these experiments place Cdk2 downstream of Notch and upstream of Mcidas in specifying MCCs. In order to identify the cellular compartment in which Cdk2 functions, tagged overexpressed Cdk2 was localized at ALI+4d, a timepoint at which both mature and ciliating MCCs are present. The authors claim nuclear exclusion of HA-Cdk2 in mature (but not ciliating) MCCs and go on to co-localize HA-Cdk2 to the centriolar compartment. In order to identify the cognate cyclin with which Cdk2 associates during MCC development, the authors utilize qRT-PCR and note the potent induction of Ccna1 during the mid-ciliogenesis stage specifically in MCCs (defined here by Foxj1 expression). Reporter assays and endogenous qRT-PCR experiments upon enforced overexpression of MCC transcription factors suggest Ccna1 can be induced by E2F4 in the presence of Mcidas. The authors next examine the localization of ectopic and native Ccna1, concluding Ccna1 is nuclear localized during ciliogenesis and subsequently undetectable or nucleocytoplasmic in mature MCCs. Given the similarity of this pattern to that previously observed for Cdk2, they argue for the function of Cdk2/Ccna1 as a physically interacting complex. In the final series of experiments the authors demonstrate the in vivo importance of Ccna1 in ciliogenesis by examining the airways of previously generated All the reviewers agree that there is broad general interest in these findings. It is clear that Cdk2/Ccna1 are important regulators of airway ciliogenesis. The data are convincing that Cdk inhibitors interfere with both processes: centriologenesis and cilia formation. This is an important contribution. However, there are substantive concerns that need to be addressed about the interpretation and mechanism and the various steps of ciliogenesis and maintenance that are affected by Cdk2.Major Concerns that must be addressed:1) The actual role of Cdk2 in ciliated cell formation, ciliogenesis, and cilia maintenance per se is unclear. They need to clarify effects on cell fate specification and ciliogenesis and ciliated cell maintenance. Is Cdk2 directing specifically the initiation and (perhaps) maintenance of MCC centriologenesis or is there a broader role in the (co-)regulation of other steps of cilia assembly. A specific role in centriole replication needs to be clear. It may be that there are biological or technical limitation such that a failure of initiation of centriole amplification halts the entire subsequent program of ciliogenesis, this may be difficult to prove in a revision. However, the apparent later stage role of Ccna1, after centriologenesis, seems contradictory to the proposal that Cdk2-Ccna1 are regulating centriole replication and needs to be resolved. There might be some experimental limitations that cannot fully separate the roles of Cdk2 in regulating ciliated cell formation and centriologenesis. But the authors need to clarify the specific roles of Cdk in either dilated cell formation or centriologenesis and also discuss these limitations in the paper2) The links between the celluar localization of Cdk2, the activation of Cdk2, and the expression of MCC TFs are inconclusive. It is difficult to appreciate the nuclear-cytoplasmic shuttling during ciliogenesis. In Figure 4, nuclear vs cytoplasmic lysates would be better than this IF (which isn't convincing for nuclear exclusion). One could immunoprecipitate tagged Cdk2 from nuclear and cytoplasmic compartments at various timepoints and show Ccna1 blots?3) The experimental evidence is not sufficient to support that CdK2 acts downstream of Notch signaling. The authors cannot exclude the possibility that Cdk2 and Notch signaling function parallel but share the same downstream consequence.Requirements for revision:1) The authors should provide additional evidence that Cdk2 acts downstream of Notch signaling, rather than in a parallel pathway. A further assessment of the components of Notch signaling might be one approach.2) The authors should provide more convincing evidence of nuclear-cytoplasmic shuttling with the HA-Cdk2, in control, basal cells and in cells undergoing centriologenesis and ciliogenesis. Identification of the fusion protein in Isolated nuclear and cytoplasmic fractions could be performed.3) Biochemical evidence of the interaction of Cdk2 with Ccna2 in cyotoplasmic fractions during specific stages of centriologenesis and/or cilia assembly. Related to this strengthen evidence regarding the relationship of HA-Cdk2 and components of centrioles .4) A better characterization of pT160 Cdk2 to identify which specific stages of MCC differentiation are associated with this activity. For example, is there coincident biochemical evidence of high levels of the massive centriole replication (and the presence of other markers of centriologenesis) or it later, post centriole replication/docking and instead coincident with cilia assembly? Or, during both processes?5) There are several relatively minor points that ought to be addressed related to clarifying the use of specific parameters in graphs, providing statistics, controls for western blots, and quantifying ciliated vs non-ciliated cells in Ccna1 ko mice.6) Finally, the last four paragraphs of the Discussion could be condensed since it is speculation and not directly focused on the data. I think that one major emphasis of the discussion should be a comparison to Al Jord et al., 2017, who show that Cdk1 (rather than Cdk2) in balance with APC is required for centriole amplification-ciliation and inhibition of mitosis, respectively. The Vlader discussion should further explain why Al Jord implicates Cdk1 and Vlader Cdk2 beyond what is written in paragraph two. The authors should discuss specifically if they believe that their data indicate that Cdk2 is directly a program or has specific protein targets, these issues are discussed loosely in paragraphs three, four and five. Consider inhibition of APC and examine Cdk2-dependent centriole amplification, this would address the discussion point in paragraph six. Major Concerns that must be addressed:1) The actual role of Cdk2 in ciliated cell formation, ciliogenesis, and cilia maintenance per se is unclear. They need to clarify effects on cell fate specification and ciliogenesis and ciliated cell maintenance. Is Cdk2 directing specifically the initiation and (perhaps) maintenance of MCC centriologenesis or is there a broader role in the (co-)regulation of other steps of cilia assembly. A specific role in centriole replication needs to be clear. It may be that there are biological or technical limitation such that a failure of initiation of centriole amplification halts the entire subsequent program of ciliogenesis, this may be difficult to prove in a revision. However, the apparent later stage role of Ccna1, after centriologenesis, seems contradictory to the proposal that Cdk2-Ccna1 are regulating centriole replication and needs to be resolved. There might be some experimental limitations that cannot fully separate the roles of Cdk2 in regulating ciliated cell formation and centriologenesis. But the authors need to clarify the specific roles of Cdk in either dilated cell formation or centriologenesis and also discuss these limitations in the paperThis is a complex multi-part question, which we attempt to break down and address systematically.First, it is apparent that we failed to adequately convey the currently accepted understanding of these events. We hope that the following is a clearer description of the events and regulation of cell fate specification, motile ciliogenesis and cilium maintenance during multiciliated cell (MCC) differentiation. To amend the manuscript, we have added two new schematics to Figure 1—figure supplement 1 , updated Figure 1—figure supplement 1C and Figure 7B (now 7C) and clarified the text in the Introduction as well as the Discussion.absence of Notch signaling that leads to the MCC fate. We note that typically, Notch signaling entails a feedback, in which neighboring cells emerge as either Notch expressing cells, in which Notch is activated , or ligand expressing cells, in which Notch is not activated . In the prospective MCCs in which Notch is not activated, it appears that MCC differentiation is a default occurrence, though we cannot rule out that some additional initiating event is required that might or might not be linked to having expressed ligand.To summarize: upon airway epithelialization, MCC and secretory cell fates are acquired in a Notch signaling-dependent mechanism. Cells in which Notch signaling is activated are diverted to the secretory cell fate, whereas cells in which Notch is not activated differentiate into MCCs. Therefore, it is the Thus, the first key point that we make is that the idea of being downstream of an ABSENCE of another event  is a bit slippery. If having expressed the ligand, or receiving another signal, is not necessarily required, one might alternately describe this scenario as the progenitors retaining a pre-existing MCC fate unless they are diverted to the secretory fate through Notch activation. Of note, published data suggest that cells diverted to the secretory fate can be transdifferentiated back to the MCC fate by inhibiting Notch, indicating that the potential to become an MCC is not eliminated, but rather is merely suppressed, by ongoing Notch signaling. Because the opportunity to divert to the secretory cell fate exists prior to MCC differentiation (see below), we choose to retain the language existing in the literature of MCC fate occurring ‘downstream’ of Notch signaling, while expressing this caveat in the amended text.in addition to a role in starting the process. This ongoing requirement may reflect a continuing transcriptional activity, but might also, or in addition, be associated with a more direct role of Cdk2 in a downstream process, a possibility suggested by the centriolar pool, for example. We suggest that investigation of the distinct pools of Cdk2 and roles other than initiating and sustaining MCC TF activity are beyond the scope of this manuscript.The second key point is that the entire morphogenetic program of MCC differentiation, including not only centriologenesis, but all the subsequent events including docking, axonemogenesis, and likely numerous other events, are driven by the activation of a MCC-specific transcriptional complex comprised of E2F4/5, Mcidas and DP1 (the EMD complex) to initiate and drive motile ciliogenesis. EMD turns on multiple other secondary transcription factors  and they cooperate to turn on hundreds of structural and regulatory ciliary genes. This leads to activation of the motile cilium biogenesis pathway, which consists of centriole amplification followed by centriole apical transport and membrane docking and then axoneme elongation. These transcription factors are expressed throughout differentiation, and most (with the exception of Foxj1 and possibly Foxn4) are downregulated only after differentiation is complete. Therefore, activating the EMD complex, a step requiring Cdk2, drives not just centriologenesis, but the entirety of the differentiation program. Ciliogenesis is a linear pathway, and a failure to initiate centriole assembly will absolutely preclude subsequent steps in the ciliogenesis program. This in itself does not distinguish between an initiation and a maintenance function for Cdk2. However, our result showing that cells in which Cdk2 is inhibited after the centriole replication has begun nonetheless are arrested in differentiation  demonstrates an ongoing requirement for Cdk2 With this conceptual framework in mind, we can address some specific queries:Is Cdk2 directing specifically the initiation and (perhaps) maintenance of MCC centriologenesis or is there a broader role in the (co-)regulation of other steps of cilia assembly.Our data support a role for Cdk2 in nascent MCCs  upstream of the EMD transcriptional complex. Because EMD drives the whole constellation of differentiation events, we propose that Cdk2 is required in nascent MCCs to initiate ciliogenesis. Our data noted above  also supports an ongoing role for Cdk2 during differentiation.However, the apparent later stage role of Ccna1, after centriologenesis, seems contradictory to the proposal that Cdk2-Ccna1 are regulating centriole replication and needs to be resolved.We do not view these roles as contradictory. Both centriole replication and subsequent events are dependent on EMD activation. As described above, both functions might be mediated by Cdk2-Ccna1 acting on EMD, or the later maintenance of ciliogenesis could potentially be mediated by non-transcriptional functions instead of, or in addition to, a transcriptional function.They need to clarify effects on cell fate specification and ciliogenesis and ciliated cell maintenance.We have added new data, requested by the reviewers, that incidentally allows us to address this question more precisely. The new data  regards the role of Cdk2 in relation to TRRAP in this process. TRRAP, a histone deacetylase complex member  was identified as an early regulator of motile ciliogenesis and was shown to act in the nucleus to turn on the EMD complex downstream of (in the absence of) the Notch signaling event. We show that TRRAP expression is induced in the absence of Cdk activity, indicating that TRAPP functions either upstream of, or in parallel to, Cdk2 activation . We allowed MTEC cultures to differentiate for four days in the presence of the Cdk inhibitor Nu6140. As shown before, these cultures are not able to initiate EMD activity and undergo motile ciliogenesis. However Nu6140 treatment had no effect on the cells’ ability to turn on nuclear TRRAP expression. Thus, we while Cdk2 is required to initiate ciliogenesis, we can conclude it is not involved in the MCC fate decision as evidenced by the expression of TRRAP even in the absence of Cdk2 activity.There might be some experimental limitations that cannot fully separate the roles of Cdk2 in regulating ciliated cell formation and centriologenesis.We presume here that the reviewer means distinguishing initiation of the differentiation program from a specific and direct function in centriologenesis. Indeed, answering this question would require biochemical analyses to complement the genetic manipulations performed thus far. As described in our response to point 2 below, the asynchrony of differentiation and the limiting amounts of material render these types of studies infeasible. In case we are interpreting the comment incorrectly, and the reviewer means separating the roles of Cdk2 in MCC cell fate specification versus centriologenesis, we argue that the TRRAP result shows that Cdk2 is not required for acquisition of the MCC cell fate.2) The links between the celluar localization of Cdk2, the activation of Cdk2, and the expression of MCC TFs are inconclusive. It is difficult to appreciate the nuclear-cytoplasmic shuttling during ciliogenesis. In Figure 4, nuclear vs cytoplasmic lysates would be better than this IF (which isn't convincing for nuclear exclusion). One could immunoprecipitate tagged Cdk2 from nuclear and cytoplasmic compartments at various timepoints and show Ccna1 blots?Two ideas appear to be embedded in this comment. First, “The links between the cellular localization of Cdk2, the activation of Cdk2, and the expression of MCC TFs are inconclusive.” An abundance of prior data establishes that Cdk2 activation and its nuclear localization (and phosphorylation) are coincident. We have shown that its activity is required for EMD complex (MCC TF) activation, and we demonstrate the correlated nuclear localization and Thr160 phosphorylation of Cdk2. These conclusions are based on inhibitor and dominant negative studies, IF assays of nuclear localization  and immunoblotting. Thus, the requirement for Cdk2 to activate EMD, and the markers of active Cdk2 are conclusive.Ccna1 we specifically show that its expression is restricted to MCCs. This means that any biochemical fractionation or IP would require purification of MCCs in the process of ciliating from a complex cell population. We have the ability to FACS sort MCCs vs. nonMCCs using the Foxj1-EGFP mouse line , but this preferentially isolates mature MCCs and cannot distinguish between MCCs at different stages of ciliogenesis . There are currently no FACS markers specifically for ciliating MCCs or specific ciliogenesis stages. Finally, even if we could sort the desired cells, it is currently not technically feasible to scale up the MTEC culture  to produce a sufficient quantity of cell lysates for the required fractionation and IP experiments. Experiments from unsorted MTECs would not address these questions or would be impossible to interpret due to the presence of nonMCCs or mature MCCs. It is already well-known from work in other cells and tissues that Cdk2 can undergo nucleo-cytoplasmic shuttling and that it can interact with Ccna1. To add additional value to our manuscript, testing this specifically in ciliating MCCs would be required, but we are unable to isolate these cells.We acknowledge that we do not demonstrate that the EMD complex (MCC TF) is a direct target of Cdk2, and that we provide only immunofluorescence evidence that Cdk2 (and Ccna1) (co)localize in the nucleus in ciliating MCCs. Technical limitations and the nature of MCC differentiation prevent us from addressing these questions biochemically. MTECs (as well as the in vivo airways) undergo MCC fate acquisition and motile ciliogenesis in a highly asynchronous manner . Although younger MTECs are more enriched for cells in the early stages of ciliogenesis and older MTECs contain mostly mature MCCs, MTECs at all times contain MCCs at different levels. Moreover, MTECs contain a large fraction of nonMCCs, which include basal stem cells and secretory cells . Our data suggest that Cdk2-Ccna1 is required for ciliogenesis, but not in mature MCCs, and for “It is difficult to appreciate the nuclear-cytoplasmic shuttling during ciliogenesis.” As we are left with only IF assays, we strengthened our observation of nuclear localization by providing careful quantitation of our Cdk2-HA localization in MTECs, the results of which support the presence of Cdk2 in the nucleus in ciliating, but not in mature MCCs .In the manuscript, we are careful to temper our overall conclusions in this revision in light of these limitations. We hope it will be possible to address these points more fully as technology advances. However, the remarkable conservation of Cdk2 localization and function  between MCCs and cycling cells complements our data and strongly supports our proposed mechanism.3) The experimental evidence is not sufficient to support that CdK2 acts downstream of Notch signaling. The authors cannot exclude the possibility that Cdk2 and Notch signaling function parallel but share the same downstream consequence.As discussed above, it is absence of Notch signaling that allows MCC differentiation. To reword the reviewer’s idea, s/he is asking whether Cdk2 might function in parallel and have common downstream consequences with the absence of Notch signaling. Said this way, the idea makes little sense. To reiterate, we argue that progenitors must both NOT have Notch activation, and have activated Cdk2. Since Notch activation suppresses MCC differentiation, temporally the Notch mediated event must occur prior to Cdk2 activation or no secretory cells would be produced. This idea is reinforced by the observation that TRRAP expression, an indicator of entering the MCC pathway (and therefore of having NOT been activated by Notch), occurs in the absence of Cdk2 activity . Thus, Notch signaling occurs before Cdk2 activation temporally, but strictly speaking, it is incorrect to say that absence of Notch signaling is upstream of Cdk2 activation.The other challenging notion in this comment is that there is a downstream consequence of not being activated by Notch. As far as we know, the consequence might be to retain the same state that the cell was in prior to or in the absence of any Notch signaling at all.Requirements for revision:1) The authors should provide additional evidence that Cdk2 acts downstream of Notch signaling, rather than in a parallel pathway. A further assessment of the components of Notch signaling might be one approach.We hope that the conceptual challenge in the idea of an activity being downstream of the absence of another signal has been adequately explained above. Strictly speaking, our data are consistent with a model in which both the absence of the Notch signal and the activation of Cdk2 are required in a given cell to promote the MCC fate. Because diversion to the secretory cell fate by Notch activity suppresses Cdk2 activation and the MCC fate, the decision about entering the secretory pathway  vs the MCC fate  must typically occur prior to activation of MCC TFs, and this (together with a lack of complete rigor) is probably the origin of the ‘upstream’ and ‘downstream’ descriptors currently in the literature. We have attempted to convey this admittedly somewhat subtle concept both here and in the revised text , but have chosen to keep the language of ‘upstream’ and ‘downstream’ as shorthand.Because it is absence of Notch signaling that is required for the MCC cell fate, investigating components of Notch signaling such as Notch intracellular domain (N-ICD) nuclear translocation and resulting gene expression would be relevant to the secretory cell rather than the MCC, and therefore not useful. We attempt to clarify the role of Notch signaling in MCC specification in the text as well as in the new Figure 1—figure supplement 1A-B.2) The authors should provide more convincing evidence of nuclear-cytoplasmic shuttling with the HA-Cdk2, in control, basal cells and in cells undergoing centriologenesis and ciliogenesis. Identification of the fusion protein in Isolated nuclear and cytoplasmic fractions could be performed.As stated above, technical limitations and the nature of MCC differentiation prevent us from addressing Cdk2-HA nuclear vs. cytoplasmic enrichment using nuclear and cytoplasmic fraction lysates. Like the in vivo airway surface, MTECs contain MCCs and nonMCCs, and MCCs ciliate asynchronously; thus a bulk extract would contain nuclear and cytoplasmic fractions from cells that would be expected to show multiple Cdk2 enrichment patterns (ciliating vs. mature cells) and thus make it impossible to interpret the results. As described above, we do not have the ability to isolate ciliating MCCs specifically due to the lack of appropriate FACS markers and the low number of these cells in our small scale primary cultures. To support our findings, we now provide quantitation of our Cdk2-HA subcellular localization in MTECs, and the results support the nuclear enrichment of Cdk2 in ciliating, but not in mature MCCs .3) Biochemical evidence of the interaction of Cdk2 with Ccna2 in cyotoplasmic fractions during specific stages of centriologenesis and/or cilia assembly. Related to this strengthen evidence regarding the relationship of HA-Cdk2 and components of centrioles .This experiment requires demonstration that Cdk2 interacts with Ccna1 (we assume that the Reviewer/Editor is asking about Ccna1 and not Ccna2) specifically in ciliating MCCs. Cdk2 is already known to physically interact with Ccna1 based on work in other systems. However, the technical limitations preclude isolation of this sub-population of cells. We raise and discuss this limitation to our study in the discussion. Our hypothesized mechanism involving a Cdk2-Ccna1 complex in early ciliogenesis regulation is bolstered by analogy to a vast trove of exiting data from other studies that indicate that Cdk2 requires A or E-type cyclin binding for activation.4) A better characterization of pT160 Cdk2 to identify which specific stages of MCC differentiation are associated with this activity. For example, is there coincident biochemical evidence of high levels of the massive centriole replication (and the presence of other markers of centrioliogenesis) or it later, post centriole replication/docking and instead coincident with cilia assembly? Or, during both processes?Again, technical limitations do not permit the isolation of the specific cells necessary for a biochemical approach to this question. It might have been possible to address this using IF if the T160 Cdk2 antibody were effective in IF experiments. Unfortunately, we were not able to obtain any immunofluorescence signal with the Cdk2 antibody using multiple fixation methods, suggesting either that the antibody does not work in IF experiments or it is not sufficiently sensitive for the amounts of phospho-Cdk2 expressed. Therefore, we are limited to inference from the increased pT160 Cdk2 signal in early ALI timepoints from bulk isolates that are strongly enriched for MCCs at the earliest stages of ciliogenesis that active Cdk2 is more abundant in these cells.5) There are several relatively minor points that ought to be addressed related to clarifying the use of specific parameters in graphs, providing statistics, controls for western blots, and quantifying ciliated vs non-ciliated cells in Ccna1 ko mice., eLife:Please note: on 5/2/18 we requested and received the following clarifications on this point by email from Maria Guerreiro, Journal Development EditorConcerning Figure 1C/Figure 4—figure supplement 1, the label on the y-axis is not described in the figure legend and is vague. Is confluence of cells binary or quantified? Is this instead confluence/density of MCC? It doesn't change after ALI is established, so how the denominator changes during different stages is unclear. Please clarify.MTECs proliferate to confluence after about 3-4 days of submerged culture. Confluence is evaluated either visually  or it can be inferred from increased transepithelial electrical resistance as measured by an epithelial voltohmmeter. We changed the y-axis labels on both figures to ALI-1d (one day before ALI). The text indicates that this corresponds to a confluent, but not yet differentiating culture. We use cDNA from this time point as a comparison for MCC-related gene expression at later time points as it reflects an intact epithelium with no MCCs.Figure 5A: the mRNA expression level of Ccna1 at the late ciliogenesis stage is higher than that at the early ciliogenesis stage. But the protein expression levels of Ccna1 in the Figure 5B show an opposite result.We speculate that at the conclusion of ciliogenesis Ccna1 protein may be actively degraded. This is supported by our inability to detect it by immunofluorescence in individual mature MCCs  and by analogy with data from other systems and processes that demonstrate that A-type cyclin levels are under strict control by gene expression as well as proteasomal degradation. MTEC lysates and cDNA preps from the three timepoints contain material from a complex mix of MCCs and nonMCCs, although the MCC fraction is enriched for cells at the early, mid and late stages of ciliogenesis. Late ciliogenesis samples also contain many mature and nearly mature MCCs, and it is possible that active degradation of Ccna1 in these cells results in the overall lower signal on the Western blot compared to the corresponding qPCR experiment. It is also possible that the cells that were used for protein lysate and cDNA prep contained a somewhat different proportion of MCCs vs. nonMCCs or ciliating (with higher Ccna1 gene and protein levels) vs. mature (with lower Ccna1 gene and protein levels) MCCs. In either case, both the qPCR and the Western blot results support a trend indicating that actively ciliating MCCs have high Ccna1 transcript and protein levels, which is downregulated as ciliogenesis concludes – supporting a role for Ccna1 in regulating this process.In addition to quantifying ciliated vs. non-ciliated cells in Ccna1ko mice and including Western blot loading controls, there are two other minor things that need to be clarified: 1. Figure 3: The authors stated that they did not detect a statistically significant difference in the Myb+/Foxj1- population under the Cdki treatment. However, in the Figure 3C, the fraction of Myb+/Foxj1- is significantly decreased (p<0.05) in the Nu6140 acute treated ALI at D4 as compared with untreated ALI at D3.Ccna1KO results is now included in the manuscript .Quantification of the Ponceau-S stained membranes serving as loading control for Western blots are now show in the following: new Figure 4—figure supplement 4B for Figure 4C and new Figure 5—figure supplement 5B for Figure 5B.We thank the Reviewers for catching this, as it is an error. We have now corrected the figure to indicate that there was no significant change detected in the Myb+/Foxj1- population in the Nu6140 acute treated ALI+4d as compared with untreated ALI+3d (t=0).6) Finally, the last four paragraphs of the Discussion could be condensed since it is speculation and not directly focused on the data. I think that one major emphasis of the discussion should be a comparison to Al Jord et al., 2017, who show that Cdk1 (rather than Cdk2) in balance with APC is required for centriole amplification-ciliation and inhibition of mitosis, respectively. The Vlader discussion should further explain why Al Jord implicates Cdk1 and Vlader Cdk2 beyond what is written in paragraph two. The authors should discuss specifically if they believe that their data indicate that Cdk2 is directly a program or has specific protein targets these issues are discussed loosely in paragraphs three, four and five. Consider inhibition of APC and examine Cdk2-dependent centriole amplification, this would address the discussion point in paragraph six.The Discussion has been condensed and we now discuss and compare in more detail our study regarding the role of Cdk2 and the Al Jord study on Cdk1. The APC inhibition experiment is interesting but we feel that it is beyond the scope of this study.


Adolescents use social media for information on medical and social aspects of maturation.The aim of this study was to investigate the concerns and information needs of adolescents regarding menarche and first sexual intercourse.Questions about menarche or first sexual intercourse were obtained from Yahoo Answers, a community-based social media question-and-answer website. A total of 1226 questions were analyzed. We focused on 123 question pairs made by users who asked questions on both topics and reported their ages at each. Quantitative and qualitative analyses were performed on these question pairs.P<.001, chi-square test). Older age at menarche was associated with greater lack of communication with parents .Qualitative analysis identified uncertainty as a significant theme for both menarche and first intercourse. Quantitative analysis showed that uncertainty was expressed in 26% (13/50) of menarche questions and 14% (7/50) of intercourse questions. Lack of communication was expressed in 4% (2/50) of menarche questions, compared with 8% (4/50) of intercourse questions. Ages at menarche and at first sexual intercourse were correlated, with women reporting menarche at the age of 13 years or younger being 2.6 times more likely to experience first sexual intercourse before the age of 16 years (The questions of adolescents on the topics of menarche and first sexual intercourse express anxiety and uncertainty and are associated with a lack of information and deficient communication with parents. The more normative and expected a behavior, the less these factors appear. Therefore, parents and educators should, to the extent possible, improve communication around these topics, especially when they occur at less typical ages. Today’s generation of adolescents is the largest in human history. With a population of 1.8 billion, 10- to 24-year-olds now comprise over a quarter of the global population. Adolescents have been a neglected age group in health care owing to various socioeconomic, political, and cultural factors that may deprive this age group of information, services, and health care professionals who are age-appropriate, gender-appropriate, nonjudgmental, and supportive . AccordiDuring adolescence, the adult self begins to emerge and one’s self-identity is born . The phyThe onset of puberty has long been accepted as the starting point of adolescence . Key socIn this study, the intersection of biology and social context in adolescent SRH is examined as it influences health-related behaviors and health outcomes. An example of a health-related behavior is engagement in unsafe sexual activity. Adolescents make up a quarter of the sexually active population in the United States and acquire half of all sexually transmitted infections . Adolescfirst period and a phrase that could indicate an age , and the second question in the pair contained the word sex and phrases that would indicate first intercourse .We extracted, using a crawler, a sample of question pairs from Yahoo Answers , a commuThe extracted questions were independently labeled by 2 of the authors such that 100 questions were labeled by both, and the remaining questions by one of the authors. Each question was labeled as to whether the first question in a pair was related to menarche and separately whether the second question was related to first intercourse. In addition, for those questions labeled in the affirmative, the labelers extracted the age of menarche or first intercourse, if the asker provided it in the question. Agreement among the raters was estimated for the 100 questions labeled by both using Cohen kappa statistic .After filtering, the questions were paired according to the anonymized user identifier provided by Yahoo Answers.These data are particularly reliable because previous studies ,11 showeThis study was approved by the Institutional Review Board of the Technion.A narrative approach was used to analyze the dataset to determine the distrust adolescent females have regarding parent-adolescent communication about menarche and first sexual intercourse. We subjected the content of all the questions and comments of each subject to a QSR NVivo analysis  to identThe data were then subjected to quantitative analysis on the variables of menarche and first intercourse.A total of 1226 questions were analyzed. Of those, 270 were judged to discuss menarche and 147 were judged to discuss first intercourse. A subset of 123 users asked questions in both the menarche and first intercourse sets and reported ages therein.P<.001). The agreement for whether the second posting was about first intercourse was 0.87 (P<.001). These values are considered very high.Cohen kappa agreement among raters for whether the first posting was menarche-related was 0.72 , but how do I tell my dad I started my period? Next month will be my 4th period, please help. Like 3 years ago (btw I’m 14 now) he got my aunt to buy me some pads and talk to me about it. Please give me hints and/ or tips on how to tell him or even your own personal stories! Also I’m really shy so I don’t feel comfortable talking to any friends moms about it! BUT PLEASE HELP ME!”“Help!!!!!!!! I'm 11 yr. 12 in August, I have no idea what to do. I told my mom, but she said it’s kind of early for me... I'm the only one in my class, who has started and how am I supposed to hide it? I just take it..Help!!! I need somebody, I feel really alone. I know if I tell one of my 3 sisters they are going to tease me and say awwwww you are growing up. I just have no idea what to do!!! OMG!”“I’m 18 and my mom made me an appointment for the gyno because I have irregular periods...at the time she made it I had never had sex before...last night was my first time...she filled out the sheet and everything saying no I’m not sexually active and all that...I’m not telling her that I had sex last night. I do not plan on having sex again anytime soon considering it was a big mistake last night. Since it was my first time and I’m not going to be doing it all the time. Now do I have to tell the gyno I have had sex? If so, should I just leave the sheet checked no and then tell them I filled that out before it happened?”P<.001). The agreement for first intercourse (compared with a study by Martinez and Abma [P=.01).A total of 270 women reported their age in their menarche question, and 147 women reported their age in their first intercourse question. The distribution of ages is shown in and Abma ) is 0.95P<.001, chi-square test). This finding is compared with literature that identifies adolescent females experiencing early menarche as twice as likely to have had intercourse by the age of 16 years than adolescent females with late menarche [P=.002).Following the themes identified in the qualitative research, we coded 50 question pairs for uncertainty about future effects, lack of information, and lack of communication with parents. Strikingly, 86% (43/50) of menarche questions and 44% (22/50) of first intercourse questions contained requests for information. Uncertainty was expressed in 26% (13/50) of menarche questions and 14% (7/50) of intercourse questions, and lack of communication was expressed in 4% (2/50) of menarche questions compared with 8% (4/50) of intercourse questions. An analysis of variance model found statistically significant interactions only between age of menarche and lack of communication  accessing SRH care may explain why nearly one-fourth of young people say that they have not discussed sexual topics with a parent . We beli


HMP) available through the R CRAN website.Regressing an outcome or dependent variable onto a set of input or independent variables allows the analyst to measure associations between the two so that changes in the outcome can be described by and predicted by changes in the inputs. While there are many ways of doing this in classical statistics, where the dependent variable has certain properties , little progress on regression where the dependent variable are microbiome taxa counts has been made that do not impose extremely strict conditions on the data. In this paper, we propose and apply a new regression model combining the Dirichlet-multinomial distribution with recursive partitioning providing a fully non-parametric regression model. This model, called DM-RPart, is applied to cytokine data and microbiome taxa count data and is applicable to any microbiome taxa count/metadata, is automatically fit, and intuitively interpretable. This is a model which can be applied to any microbiome or other compositional data and software (R package  If the composition of the taxa population is different for changing levels of cytokines, these can be examined as a possible mechanism of disease or for treatment targets4.Researchers may want to know how microbiome composition (outcome or dependent variable) changes in relation to patient characteristics (inputs or independent variables) such as age, disease severity, or cytokine level. The value of this type of analysis is to understand how patient characteristics may directly impact microbial compositional and variability. For example, if we know the microbiome changes in a systematic way as patients age, those changes due to age can be accounted for and removed when studying disease processes and the differences between healthy and sick patients5. As computing power increased over the years, new regression algorithms have emerged that apply to more complex models and outcomes. These include nonlinear, mixed, and nonparametric regression  models6, and nonparametric recursive partitioning to finds regions of the covariate space where the outcome is more homogeneous8. Within the recursive partitioning framework even more complex outcomes have been modeled such as survival models9 or Haseman-Elston models from genetic epidemiology to associate genetic markers in sibpairs with disease10.Associating patient outcomes (dependent variable) with input covariates (independent variables) is often a regression analysis problem to statisticians. Regression methods from classical statistics have been developed such as linear regression for numerical outcomes like cholesterol levels, logistic regression for binary outcomes like responder versus non-responder, or proportional hazards regression for survival analyses like time to deathad hoc regression methods. For example, we regressed individual taxa proportions  onto day of life, days on antibiotics, breast milk, and C-section in a study of necrotizing enterocolitis in pre-term births to see how these variables impacted the levels of these taxa11. Since each taxon was regressed separately, this approach was suboptimal by not using the covariation information among the taxa and metadata in the analysis, and it was not possible to model how all three taxa changed simultaneously. A second approach uses random forests, however these models use the individual taxa as inputs or independent variables to predict an outcome or dependent variable, such as a source of pollution12. While this is an important question, it is addressing the inverse problem of how does the microbiome composition change as vales of different meta-data (inputs) change. It also treats each taxon separately ignoring the information contained in the covariance structure of the taxa. A third approach is to cluster samples using distance measures calculated from the microbiome data and compare the metadata distributions across subgroups, such as done to detect differences in microbiome composition between non-demented and demented patients13. The weakness of this approach is that the clustering and analysis metadata are independent and not using the joint information inherent in the multivariate taxa count/metadata data resulting in a less powerful statistical analysis.However, these models won’t work off-the-shelf when the outcome (dependent variable) are the taxa counts and the inputs (independent variables) are the metadata since the mathematics and computing behind them don’t apply to microbiome taxa count outcomes. To get around this, researchers have developed 14. However, the sparseness of the taxa count data makes estimating regression coefficients and error terms difficult, and often these cannot be estimated.In regression the analysis of microbiome compositions and metadata should be done jointly to make full use of the covariance information in the data. Further, we are interested in ‘regressing microbiome data onto metadata’ which identifies the taxa counts as the outcome or dependent variable, and the metadata as the input or independent data. One attempt to regress microbiome data onto metadata has been previously proposed15, and the well-established nonparametric recursive partitioning regression framework16. The Dirichlet-multinomial recursive partitioning model, DM-RPart, takes the taxa compositions as the output or dependent variable, and the metadata as the inputs and finds partitions of the metadata where the microbiome samples differ. The following sections describe the algorithm and give an example showing how different cytokine values result in different microbiome composition. DM-RPart is available in the R HMP package as open source code through CRAN. In addition, the data and code to generate Fig. Regression, when the outcomes are microbiome taxa counts or compositions, will allow investigators to predict how taxa counts change for different values of covariates. In this paper we propose a novel and practical approach for regressing microbiome data onto metadata that analyzes the data jointly, is automatic and computationally easy, and is intuitively interpretable. This method combines the Dirichlet-multinomial probability model which has been shown to apply to microbiome compositional data17. The Dirichlet-multinomial recursive partitioning algorithm (DM-RPart) allows the regression of the compositional data onto the covariates without having to resort to one-taxa-at-a-time, complex assumptions, or data reduction.The method presented here has been developed for microbiome compositional data outcomes using recursive partitioning that separates samples into non-overlapping subgroups based on patient covariate values – samples within a subgroup are more similar to each other in their microbial composition than to samples in other subgroups. Similarity of a separation into two subgroups is measured by the Dirichlet-multinomial (DM) model loglikelihood ratio19. Twelve cytokines were selected that may represent one or more physiological process associated with antimicrobial activity 20, autoimmunity (Eotaxin)21, allergy 22, and viral infection (IFNG)23, as well as cytokines related to obesity (Leptin)24 and regulation of inflammation (TGFB)25. Partitioning all 128 microbiome samples produced an optimal tree that first split samples based on leptin, then partitioned high-leptin microbiomes based on IFNG, and low-leptin microbiomes based on IL4/IL23  164(1U54DE02378901)L23 Fig. . Specifi24, and obesity has frequently been associated with changes in the composition of the gut microbiome. Interestingly, changes in leptin levels have also been associated with changes in the composition of CD4 + T cells. Increased leptin correlates with an increased Th1: Th2 ratio27, which may explain why we observe IFNG-related variation in the microbiome4 on a high-leptin background. By contrast, low leptin correlates with a decreased Th1:Th2 ratio, which may in turn explain why we observe IL4-related variation in the microbiome28 on a low-leptin background. The presence of IL23 partitioning in our model potentially indicates Th17 related variation in the gut microbiome29 may also be observed on a low leptin background. Our results therefore support an established relationship between obesity and the microbiome and, furthermore, allow us to generate novel hypotheses about leptin-dependent detection of CD4 +T cell interaction with the microbiome.This model makes biological sense. Leptin levels are associated with BMIchildren nodes based on the subjects Leptin value. Finding the cut point is described in the technical paper, but in brief, all cut points over all covariates  are tested. The covariate/cut point that splits the samples into two groups is selected and entered into the tree  is split into two ree Fig.  that resright-sized tree after the first step of fitting a full tree partitioned down to a small number of samples within each terminal node. To avoid overfitting the data, the full tree is pruned back to a smaller right-sized tree by cross-validation and cost-complexity pruning. Cross-validation error is defined as the total squared Euclidean distance between validation data to the mean taxa of each terminal node for a given subtree which is known as the PRESS statistic (see details in Methods section) which becomes smaller as the partition finds homogenous subgroups 7. The cross-validation results for the Leptin/IFNG/IL4/IL23 tree is shown in Table right-sized tree is selected as smallest mean cross-validation level which is five on the full iHMP data.The second step of the DM-RPart algorithm is to find the We subsequently used DM-RPart to examine whether insulin sensitivity may influence host-microbiome interactions. We combined samples for 27 insulin resistant (IR) and 64 insulin sensitive (IS) based on steady state plasma glucose (SSPG) measurements. The same variables as above plus a categorical variable indicating if the sample was from an IR or IS subject were included. Notably, DM-RPart returned no significant tree when partitioning microbiomes from both IR and IS individuals which are shown in Table ). For each simulation three microbiome datasets were generated using the Dirichlet-multinomial parameters Dirichlet.multinomial in the R package HMP.In this simulation we assess the performance of DM-RPart under a range of sample size (number of microbiome samples), difference between DM parameters (es Table . For eacright-sized pruned trees. The mean squared error is based on the distance of the samples in the node from the node mean (π) with the smaller distance indicates better fit of the data. Note that in the simulation, the group membership was never used for fitting the tree. Table θ increases from 0.08 to 0.6), as the sample size decreases (N from 360 to 120), and as the covariate standard deviation increases (SD from 0.2 to 0.5).The statistical assessment of DM-RPart is defined as the average of mean squared error in each terminal node for the 15 and is extended here for microbiome regression. This paper introduces a novel approach of applying recursive partitioning to find covariates that separate and explain differences in microbiome taxa count data. The utility of this approach lies in discovering covariates associated with the microbiome taxa counts, and for forming hypotheses for future testing and patient stratification in clinical trials.The Dirichlet-multinomial distribution was introduced previously for formal hypothesis testing in microbiome research30. The Dirichlet-multinomial distribution analyzes microbiome compositional data using taxa counts avoiding the need to rarefy which is known to cause loss of information31, the reduction of multivariate taxa counts into a single number diversity measure which is difficult to interpret32, and the fallacy that systems biology can be learned by univariate statistics through one-taxa-at-a-time analyses33.DM-RPart uses recursive partitioning to regress microbiome compositional data onto covariates. Microbiome taxa count data is compositional since a fixed number of sequence reads are sampled and an increase in one taxa must be accounted for by a decrease in one or more other taxa to ensure that the sum of the proportions always adds to 1Any covariates can be used for fitting trees such as treatment or disease group, age, gender, or metabolites. In the first example we focused on cytokines in order to develop hypotheses about microbiome/cytokine associations. In the second example we were interested in whether insulin resistant versus insulin sensitive patients had different microbiomes.35.The selection of the covariates is not limited, though we encourage covariate sets be selected that can be interpreted biologically versus adding hundreds or thousands of covariates that will be difficult to understand and result in spurious results due to the high dimensionality of the dataTwo known limitations of recursive partitioning are the tendency to overfit the data, and binarizing of continuous variables at each split of the tree into two levels. We address the overfitting problem using a cross-validation cost-complexity pruning algorithm. The issue of splitting continuous variables (binarizing) has been argued as a reason to avoid recursive partitioning algorithms. Nevertheless, RP is a commonly used exploratory analysis tool in the statistics community, and DM-RPart clearly provides insights into the relationship between covariates and the microbiome that cannot be quantified by other approaches. As an exploratory tool it also needs to be kept in mind that the results should be used to advise on the hypothesis testing and design of future studies. We believe the problem of binarizing covariates is outweighed by the insight this model provides.36 or multiple co-inertia analysis (MCIA)37. However, these methods differ from DM-RPart in terms of the type of outcome  or how the results are presented . Because these methods and DM-RPart are using different data and approaches a benchmarking comparison was not felt appropriate.Other methods are used in microbiome research to associate microbiome data with covariates such as PERMANOVAModels fit using the DM-RPart function in R can be used to predict the terminal node assignment and microbiome for new data with the same covariates. The ability to predict in R can be used to validate the models as well and provide independent test data estimates of accuracy.et al.15.The Dirichlet Multinomial (DM) probability distribution on the set of microbiome taxa count samples is defined as:likelihood function of the observed microbiome samples In DM-RPart, we use the likelihood ratio statistic from the DM model to partition the covariate space into non-overlapping homogeneous subregions. Let likelihood ratio test is routinely used for testing alternative models, and was used to test if two sets of microbiome samples come from the same DM model or from different DM models15. Formally, the likelihood ratio test (LRT) of the hypothesis, In classical statistics a Instead of hypothesis testing to calculate a P value, in the DM-RPart algorithm LR is used as a measure of how similar and dissimilar subsets of microbiome samples are to each other. In practice for computational accuracy, the log-likelihood Z = 1 with probability = 0.95 if Y ≤ 0.5, probability = 0.05 if Y > 0.5, X ≤ 0.5, and probability = 0.70 if Y > 0.5, X > 0.5. Otherwise Z = 0. The simulated data is shown Fig. The general framework of recursive partitioning for a binary (Yes/No) outcome is introduced to illustrate the method. A dataset with N = 100 random observations was generated with two predictor (independent) variables X and Y simulated from a Uniform distribution between 0 and 1, and a binary outcome (dependent) variable best score is selected and the data in a (parent) node are partitioned into two non-overlapping (child) nodes.The goal of recursive partitioning is to automatically find boundaries such as those in Fig. tree structure with the top node containing all the data, and the terminal nodes containing subregions of the covariate space defined by splits along the covariates.The algorithm proceeds recursively by repeating this step on each child node until all the Z (outcome) variables within a node have the same value, or there are less than some minimal number of observations need for splitting, say 5. The order of the splits defines a parent node would be classified as 1 with a misclassification rate of 35% . The first split occurs at child node, and child node. The samples in the right child node were not further split with high homogeneity of 38 class 1 samples and 1 class 0 sample for a misclassification rate of 1 out of 39, or 2.5%. The samples in the left child node were classified by majority rule as class 0 since 34 out of 61 were class 0, incorrectly classifying the 27 class 1 samples as class 0 samples, a 44.3% misclassification rate. This node was further split along child node, and child node, with misclassification rates of 2 out of 26 (7.7%) and 10 out of 25 (40.0%), respectively  of the tree16. Let children nodes into a higher parent node. The complexity of any tree, To determine the The cost complexity for any subtree For a categorical response, the relative error is calculated based on the misclassification rate for each split, with entclass1pt{minimaAn alternative method of selecting the right size tree is the 1 SE rule. Sometimes the cross-validation errors rapidly decrease for the first splits, followed by a long flat tail with small random fluctuations. When the position of the minimum in the flat tail is unstable, the 1 SE rule provides the simplest tree whose accuracy is comparable to the tree with the smallest cross-validation estimate of error rate. The 1 SE rule is implemented as follows: First define the tree 39 and genetic epidemiology10. Most standard statistical packages now contain versions of recursive partitioning.We presented recursive partitioning using a simple example with a binary outcome to illustrate the method and introduce the mathematics. This method can be used with categorical or continuous outcome variables, and has been extended to more complex models such as linear regressionDirichlet-multinomial log-likelihood ratio (LLR) and cross-validation cost-complexity pruning to avoid overfitting. The goal is to find partitions of the covariate space by recursive partitioning so microbiome samples within terminal nodes are more similar to each other than they are to microbiome samples in other terminal nodes.In this section, microbiome recursive partitioning is presented that extends classical recursive partitioning to microbiome taxa counts as the outcome variable with splitting based on the Nm covariates parent node A. For covariate child node, and all others into the right child node. As in standard recursive partitioning, the process is repeated for all cut points and covariates, with each split of a parent node scored by the loglikelihood ratio parent node, the split with the largest absolute value of child node to fit Consider a set of taxa count data on LL over all terminal nodes in the subtree The cost complexity in DM-RP for any subtree Fit Randomly partition the data into 10 equal sized subsets.th subset (Di) as the validation datasetSet the iFit the Obtain the pruned trees of i at each Compute the predicted class for all observations in Di to For each pruned tree, calculate the cross-validation error as the mean of squared Euclidean distances from DFor each subset Repeat step 3 for all Claculated the averaged cross-validation error at each Repeat step 2–5 The subtree with the smallest averaged cross-validation error of step 6 or follows the 1 SE rule is selected as the right sized treeA 10-fold cross-validation method for selecting the right sized tree is developed. In any node we can calculate the MOM estimate of the Dirichlet-multinomial parameter (I). Participants provided informed written consent for the study under a research study protocol #23602 approved by the Stanford University Institutional Review Board. (II). All research was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants and/or their legal guardians.