In 2019, a novel severe acute respiratory syndrome (SARS-CoV-2 (COVID-19)) caused a global pandemic. There was an urgent need to develop a vaccine against COVID-19 to reduce its spread and economic burden. The main objective of this study was to understand the attitudes and concerns of healthcare workers (HCWs) towards the upcoming COVID-19 vaccine, whether their decision was influenced by their history of taking the seasonal influenza vaccine, and factors that influence the acceptance of the upcoming COVID-19 vaccine. This was a cross-sectional study conducted in Riyadh, Saudi Arabia. We selected and surveyed 356 HCWs via an electronic self-administered questionnaire. A total of 61.16% of HCWs were willing to receive the COVID-19 vaccine, and 55.9% of them had received the seasonal influenza vaccine in the preceding year (2019–2020). The strongest predictors for taking the COVID-19 vaccine were the HCWs’ belief that the COVID-19 vaccine would be safe, needed even for healthy people, that all HCWs should be vaccinated against COVID-19, and that HCWs will have time to take the vaccine. Being female, being middle aged, having <5 years of work experience, having no fear of injections, and being a non-smoker were predictive factors for taking the upcoming COVID-19 vaccine. No associations were found between the intention to take the COVID-19 vaccine and a history of taking the seasonal influenza vaccine. In early 2020, a novel strain of the coronavirus, SARS-CoV-2 COVID-19), spread rapidly over the world, and the World Health Organization (WHO) declared a global pandemic in March 2020. COVID-19 is caused by severe acute respiratory syndrome (SARS-CoV-2) 9, spread; in SaudThe current global strategy to control the COVID-19 pandemic consists of suppression measures, including travel restrictions, quarantine, social distancing, case tracing, and case identification to prevent the spread of the infection . GloballVaccines are the most effective measure to control infectious diseases and reduce the economic burden . In 2009Candidate vaccines included an inactivated virus, nucleic acid, adenovirus base, and recombinant subunit vaccines . In 2019p < 0.01), and 75% of the general population [HCWs are great public influencers. Therefore, a study of their attitudes and concerns towards the COVID-19 vaccine is an accurate predictor of the general population’s acceptance of the upcoming vaccine . Congo’spulation . A recenpulation . Thus, wThe main objective of this study was to understand the attitudes and concerns of HCWs towards the upcoming COVID-19 vaccine and whether their intention was affected by their history of taking the seasonal influenza vaccine. The secondary objective was to study the factors influencing the acceptance of the upcoming vaccine.This was an analytical cross-sectional observational study. The target population was HCWs in the main hospitals of Riyadh, the capital of Saudi Arabia. Riyadh had one of the highest rates of confirmed COVID-19 cases in Saudi Arabia . Data foA validated questionnaire , which cp-values < 0.05 were considered statistically significant and a 95% CI was considered for the result. A chi-squared test was used to compare the variables and the willingness to accept the COVID-19 vaccine.Data were analyzed using Statistical Package for the Social Sciences (SPSS) version 27.0 . The sample-size estimate was based on a previous study about respondents who intended to receive the pH1N1 vaccine . Therefop-value were calculated. Multivariate logistic regression analysis was used to predict the factors influencing acceptance of the COVID-19 vaccine among HCWs. Two independent regression models were generated to assess the ability of the attitude-scale items to predict the HCWs’ willingness to accept the COVID-19 vaccine. All factors found to be statistically significant (p < 0.05) were included in the final models. The base model included all the significant factors from the sociodemographic and influenza vaccine-history sections; however, it excluded the COVID-19 attitude scale items. The base model and the COVID-19 attitude scale items include the same factors from the base model, in addition to the significant items from the COVID-19 attitude scale. The area under the receiver operating curve (AUC) was used to assess the discriminative power of both models to accurately predict the HCWs’ COVID-19 vaccination decision.Using the COVID-19 Vaccine Attitudes Scale (34 items) , univariOnly 329/411 (80%) participants completed the entire questionnaire, and 363/411 (88.3%) participants answered the question about willingness to accept the upcoming COVID-19 vaccine. Overall, 222 (61.2%) HCWs were willing to accept the COVID-19 vaccine, with almost equal agreement between men (48.2%) and women (51.8%). The participants willing to accept the COVID-19 vaccine were aged 30–49 years (51.4%) and had <5 years work experience (41.0%). The participants willing to accept the vaccine were those with no fear of injections (70.7%) and no history of smoking (85.6%). The characteristics of the participants are presented in No statistically significant relationships were found between the willingness to accept the upcoming COVID-19 vaccine and nationality, social status, history of chronic diseases, household members , having close contact with an elderly person, pregnancy or breastfeeding, location, occupation, subspecialty, work status, having direct contact with patients in general, and dealing with confirmed COVID-19 patients. The least statistically significant characteristics were subspecialty, occupation, being pregnant, and a history of chronic diseases . No statThe last seasonal influenza vaccine (2019–2020) was taken by 199 (55%) participants; only 56 (15.4%) participants took the seasonal influenza vaccine annually, while 68 (18.7%) participants had not taken the seasonal influenza vaccine in the past five years. The majority of study participants did not experience adverse effects 230/363 (63.3%) or allergies 308/363 (84.8%) from the seasonal influenza vaccine.From the participants willing to accept the upcoming COVID-19 vaccine, 55.9% had taken the seasonal influenza vaccine in the preceding year (2019–2020). None of the items assessing the history of the seasonal influenza vaccine and COVID-19 infection showed a statistically significant relationship with the willingness to take the upcoming COVID-19 vaccine .p < 0.05) and showed higher odds in those willing to accept the upcoming vaccine ; therefore, the base model predicted 68% of HCWs will accept the COVID-19 vaccine.The second model integrates the base model and the COVID-19 Vaccine Attitudes Scale to determine whether this integration will increase the ability for predicting the HCWs’ acceptance of the vaccine. As presented in To the best of our knowledge, this is one of the few studies that has explored the attitudes, concerns, and predictive factors for COVID-19 vaccine uptake among HCWs in relation to their exposure to the seasonal influenza vaccine. There were 61.2% of HCWs willing to receive the COVID-19 vaccine. However, no associations were found between the intention to accept the COVID-19 vaccine and the history of receiving the seasonal influenza vaccine.The strongest predictive factor for vaccine acceptance was the belief that all HCWs should be vaccinated against COVID-19. Vaccine acceptance was higher in women, non-smokers, participants aged 30–49 years, and those without chronic diseases. Sociodemographic factors can determine not only the HCWs’ attitude but can also influence receiving the COVID-19 vaccine . HCWs anHowever, these findings differ from another report, where the highest vaccine acceptance rates were in older HCWs and patients with chronic diseases . As our Our findings suggest that employment within the different healthcare professions  does not significantly influence the respondents’ acceptance or rejection of the upcoming COVID-19 vaccine. However, consistent with previous studies, our findings showed greater vaccine hesitation among nurses than physicians . A studyA recent national unpublished study assessed the acceptance rate of the COVID-19 vaccine among HCWs  and repoSeveral studies indicate that a positive attitude towards the seasonal influenza vaccination is a strong determinant for accepting the COVID-19 vaccine ,22. NeveWe expect that the current COVID-19 pandemic will promote a higher compliance of the seasonal influenza vaccines in HCWs, forming a bidirectional relationship ,33. The COVID-19 vaccine is the only effective measure to confine the pandemic and eventually resume much of regular life, and HCWs have priority to be vaccinated. Our study found that the COVID-19 vaccine acceptance was related to the HCWs’ modifiable beliefs and attitudes that the COVID-19 vaccine is safe, needed even for healthy people, that all HCWs should be vaccinated against COVID-19, and that HCWs will have time to take the vaccine. Research on the pH1N1 vaccine showed findings similar to our research, where HCWs were more likely to accept the pH1N1 vaccination if they perceived a susceptibility to the infection, severity of the infection, the benefits of the vaccination outweighed the barriers, and HCWs were influenced by positive external cues to action .The belief that all HCWs should be vaccinated against COVID-19 was the strongest predictive factor for vaccine uptake. This finding reflects the importance of HCWs to be vaccinated, as they are the first line of defense against COVID-19. HCWs have a critical role in promoting vaccine culture in their community. Therefore, the first step to vaccine acceptance is developing the confidence in HCWs in receiving the vaccine and recommending it to their patients .The Saudi government linked completing the vaccines by individuals to one of the mega events held yearly in Makkah, “the Hajj”. During the current holy month (Ramadan), the Saudi Arabian Ministry of Hajj and Umrah allowed pilgrims into the Grand Mosque in Makkah if they could show evidence of immunity by providing a certificate of either having received at least one dose of the COVID-19 vaccine or having a history of a previous COVID-19 infection, and this was to be presented before entering the holy mosque, praying, or performing Umrah . A healtHCWs were in the first priority group for vaccination, and they were at the first stage of the Centers for Disease Control and Prevention (CDC) COVID-19 Vaccine Rollout Recommendation as they are the frontline in fighting the pandemic . The finThis study has numerous strengths. It was conducted in Riyadh, the capital and largest city in Saudi Arabia. HCWs from multiple centers were enrolled in the study, including government hospitals, university hospitals, and private hospitals. An adequate sample size was achieved with a good response rate, despite being an online survey. Despite having an adequate sample size, we had expected a higher response rate. Online surveys are usually associated with low response rate compared with face-to face survey. This was expected especially true during the COVID-19 pandemic as many people, especially HCWs, experienced higher psychological stress, in addition to the increased number of online surveys leading to survey fatigue.This is one of the few studies that has assessed the association between the influenza vaccine status and the rate of the COVID-19 vaccine acceptance, measured the HCWs’ willingness to take the COVID-19 vaccine using an attitude scale, and tested it by multivariate logistic regression.However, the single geographical location may limit its generalizability. Data were collected before the availability of a vaccine, which could influence the HCWs’ decisions. As it was an online questionnaire open to all HCWs, it was difficult to have an equal distribution of age, occupation, and specialties in the participants.Our results recommend that the HCW attitudes and concerns towards COVID-19 should be addressed. The opinion of HCWs has high value in communities; therefore, it is essential to target HCWs with awareness campaigns to change their perceptions. Future vaccine campaigns will have a greater impact in pandemic and non-pandemic times if vaccine culture is promoted in the workplace among colleagues with the supervisor’s encouragement. The pandemic lockdown caused some difficulties in reaching the targeted sample size; therefore, we recommend establishing an electronic network to connect HCWs from different facilities and specialties. We suggest conducting further studies to assess the COVID-19 vaccine uptake after the vaccine is available. National longitudinal studies are required to assess the attitudes of HCWs towards influenza vaccines using the Vaccine Attitude Scale, as well as to assess the influenza vaccine uptake after the COVID-19 pandemic.In conclusion, most of the HCWs were willing to accept the COVID-19 vaccine. The intention of accepting the COVID-19 vaccine was not associated with previous exposure to the seasonal influenza vaccine. The modifiable factors, which were the strongest predictors for taking the COVID-19 vaccine, were the HCWs’ beliefs that the COVID-19 vaccine will be safe, needed even if the person is healthy, that all HCWs should be vaccinated against COVID-19, and that the HCWs will have time to take the vaccine.


Desmoid-type fibromatosis (DTF) is an uncommon soft-tissue tumour that is incapable of metastasising. Patients can experience a variety of physical and psychological symptoms, making assessment of health-related quality of life (HRQoL) highly relevant. Measuring the impact of DTF on HRQoL can be challenging due to the rare character and variable clinical presentation of the disease. Therefore, a HRQoL instrument assessing DTF-specific issues is needed. Previously, a provisional DTF-specific HRQoL tool was developed (the DTF-QoL). The aim of the current study was to pre-test the DTF-QoL. Pre-testing of the questionnaire led to the selection of 96 questions, conceptualised into three symptom subscales, eleven disease-impact subscales and six single items, together forming the final DTF-QoL. This questionnaire can be used in both clinical and research settings to evaluate HRQoL of DTF patients, which could help to provide personalised care and improve overall patient experience.Sporadic desmoid-type fibromatosis (DTF) is a rare, non-metastasising soft-tissue tumour. Patients can experience a variety of disease-specific issues related to the unpredictable clinical course and aggressiveness of DTF, which negatively impacts health-related quality of life (HRQoL). These DTF-specific issues are not captured by generic HRQoL tools. A 102-item provisional DTF-specific HRQoL tool, the DTF-QoL, was previously developed. The aim of this study was to pre-test the psychometric properties of the DTF-QoL by administering it together with the EORTC Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) to 236 DTF patients from the United Kingdom and the Netherlands. Construct validity and reliability were determined based on factor analysis, multi-trait scaling analysis, Cronbach’s alpha, and correlations with the EORTC QLQ-C30 scales. Ninety-six items were selected, conceptualised into three symptom scales, eleven disease-impact scales and six single items, together forming the final DTF-QoL. Scaling assumptions were fully or moderately met for ten out of fourteen scales. Cronbach’s alpha ranged from 0.551–0.908. Most scales of the DTF-QoL were weakly or moderately correlated with the EORTC QLQ-C30. The DTF-QoL is a promising tool capturing the whole spectrum of DTF-specific issues. Implementation of the DTF-QoL in research and clinical practice will help to personalise HRQoL measurement and clinical care for DTF patients. CTNNB1) gene [Desmoid-type fibromatosis (DTF) is a rare soft-tissue tumour . The estB1) gene . SporadiB1) gene . The bioB1) gene ,7.The unpredictable clinical course of DTF makes it challenging to choose an appropriate treatment strategy. Active surveillance is currently recommended as first-line treatment for asymptomatic and mildly symptomatic patients, while in the case of persistent progression or increasing symptoms, systemic therapies, surgical resection, and local therapies, such as radiotherapy, can be considered . With hiHRQoL is a multidimensional concept that includes the patient’s perception of the impact of their disease and treatment on physical, psychological, and social functioning . It provThe DTF-QoL and the EORTC QLQ-C30 were completed by 236 DTF patients (response rate 47%). Socio-demographic and clinical characteristics are described in Response information is provided in The suitability of the data (remaining 96 items) for factor analyses was tested via the Kaiser–Meyer–Olkin (KMO) measure of sampling adequacy, which tests the partial correlations among the items. The KMO value should be higher than 0.5 for a satisfactory analysis to proceed . The KMOFactor analyses for items with time frame ‘since your diagnosis’ demonstrated eleven factors cumulatively accounting for 50% of variation in all items . Oblique rotation generates a pattern matrix with factor loadings  and a stThe multi-trait scaling analysis showed that scaling assumptions were fully met for three of the fourteen scales. Seven of the fourteen scales showed that scaling assumptions were moderately met, and four scales poorly met the scaling assumptions . Cronbacr ≤ 0.60) with the EORTC QLQ-C30  and scale 5  of the DTF-QoL were strongly correlated with the physical functioning scale of the EORTC QLQ-C30 . Most sc QLQ-C30 .The final version of the DTF-QoL consisted of 96 items .The DTF-QoL has been developed according to the robust guidelines of the EORTC Quality of Life Group to measure disease-specific and treatment-related HRQoL issues relevant to DTF patients that are not adequately covered by the EORTC QLQ-C30 . The DTFExploratory factor analysis of the DTF-QoL yielded fourteen factors, which were categorised in three symptom scales (items with time frame during the past week) and eleven disease impact scales (items with time frame since diagnosis). These scales covered all the DTF-specific issues found in previous studies ,15,16. RBased on the content validity of an item with its scale, or improvement of the Cronbach’s alpha coefficient if a certain item was deleted, we explored whether rearrangement of some items would improve the psychometric properties of the scales. For scale 3 , item 78 ‘Have you experienced a lack of continuity  in the care for your desmoid fibromatosis?’ showed a weak correlation with its hypothesised scale, but had no higher correlation with another scale. In addition, removing item 46 ‘Have you felt supported by your family members and/or friends?’ from scale 4 (‘effect of DTF on relationships’) improved the reliability of scale 4. The fact that item 46 might be more person-specific rather than DTF-specific might explain why this item did not fit scale 4. Furthermore, both items 78 and 46 reflected a more generic aspect related to support, which was associated with almost all scales. For this reason, treating item 46 and 78 as single items could be considered. Moving item 52 ‘Have you felt a change in sensation in the area around the tumour?’ from scale 8 (‘body image and sensation’) to 10 (‘concerns around treatment and its consequences’) improved the Cronbach’s alpha coefficients, as well as the scaling fulfilment of both scales. Based on these results and good content validity, we recommend to move item 52 from scale 8 to scale 10. Finally, item 68 ‘Have you wanted to meet others with desmoid fibromatosis?’ of scale 9 (‘supportive care’) was formulated in a neutral way, so patients could have interpreted it positively or negatively, possibly affecting the reliability of the scale. The fact that Cronbach’s alpha was negative reflected the incoherence of item 68 with its scale. We chose not to consider the items of scale 9 as a single item directly, but suggest formulating item 68 in a positive direction and re-evaluating the psychometric properties of this scale. However, all our recommendations, as well as our hypothesised scale structure, require additional confirmatory analysis in a larger  study.Up until now, one disease-specific HRQoL instrument, the GODDESS, has been developed for DTF patients . The GODFinally, the DTF-QoL consists of a relatively large number of items compared to the GODDESS, and may be exhausting to patients. However, all items were considered to be of clinical relevance, as these issues were rated as important by patients and healthcare providers during phase I of the development process. In particular, the impact scales of the DTF-QoL include a variety of DTF-specific HRQoL issues not covered by existing questionnaires, such as the unpredictable disease trajectory of DTF or the effect of DTF on relationships. The weak correlation of most scales with the EORTC QLQ-C30 confirms divergent validity and supports the fact that the DTF-QoL identifies issues not covered by generic or cancer-generic questionnaires. Known-group comparisons will be performed next to assess the ability of the DTF-QoL to discriminate between subgroups of DTF patients with different disease phase or treatment modalities. Furthermore, based on the results of these subgroup analyses, relevant and irrelevant issues for specific subgroups will be identified. Ultimately, we hope to identify optional and essential scales for specific subgroups or even for individual patients. Furthermore, the use of different time frames offers the possibility of using a specific selection of subscales for a specific type of research. For example, the symptom scales with a relatively short time frame (past week) are appropriate for assessing a patient’s experience during a clinical trial, while the disease impact scales can potentially be used during longitudinal follow-up. Given the large number of items, which together form an extensive item library, it might be worthwhile to explore the possibilities of applying computer adaptive testing (CAT) techniques . CAT tecTo develop the DTF-QoL, the EORTC guidelines for developing questionnaire modules were followed as much as possible, however our questionnaire is not an EORTC product . The devn = 80; Netherlands (NL): n = 156) who participated in the QUALIFIED study : NCT04289077) [The sample included 236 patients (United Kingdom (UK): 4289077) . The QUA4289077) . Sociode4289077) .Patient characteristics were summarised using descriptive statistics. For phase III, item descriptive statistics and response distributions for each item were calculated, in order to examine central tendency, variability and symmetry. Floor or ceiling effects were considered high if >85% of answers fell in the lowest or highest category respectively .To support construct validity, exploratory factor analysis was used to determine underlying constructs that explained significant portions of the variance. The factor loadings, i.e., the correlation coefficients between the items and the factors, were examined in order to explain the meaning of each construct. Items deemed inappropriate for the identified scales due to their content were treated as single items. Scores of the identified scales were calculated according to the guidelines of the EORTC Quality of life group .Multi-trait scaling analysis was performed to confirm the hypothesised scale structure of the questionnaire . To testReliability was assessed by calculating Cronbach’s alpha coefficient (ranges between 0 and 1). A minimum score of 0.70 is preferred . Substanr ≥ 0.40), while scales with less conceptual relation were expected to correlate weakly (r < 0.40) [Finally, Pearson’s correlation coefficients were calculated between the scales of the EORTC QLQ-C30 and the identified scales of the DTF-QOL to assess convergent and divergent validity. Scales conceptually related were sought to show moderate to high correlations ( < 0.40) .Missing answers for patients completing the questionnaires were reported where applicable, and only available data were used for further analyses. All analyses were performed using SPSS software, version 25.0 .The DTF-QoL is the first DTF-specific questionnaire developed using the EORTC guidelines for developing a questionnaire. This questionnaire can be a useful HRQoL instrument in future  clinical studies and clinical care. It could help healthcare professionals to understand patient priorities and to recognise HRQoL issues earlier to provide truly personalised care and improve overall patient experience.


C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the  In brains, neuronal processes or neurites are segregated away from cell bodies into synapse-rich regions termed neuropils: dense structures of nerve cell extensions which commingle to form functional circuits . In bothC. elegans nerve bundles, neurite position is established and maintained via combinatorial, cell-specific expression of CAMs which mediate local neurite interactions and, when altered, lead to defects in neurite order within bundles  in undifferentiated cells from early embryos can drive their compartmentalization . This co sorting . While dneurites , where cC. elegans nerve ring. The C. elegans nerve ring is a layered neuropil, with specific layers or strata functionally segregating sensory information and motor outputs . The two AIB neurites then circumnavigate the nerve ring at opposite sides of the neuropil - both AIBL and AIBR project dorsally along the posterior neighborhood, on the left and right-hand sides of the worm, respectively . SimultaAfter meeting at the dorsal midline, instead of making a shift to the anterior neighborhood  . These vwhere borhoods . Since tTime lapse images and measurements of the developmental dynamics showed that zippering and unzippering takes place concurrently: zippering on to the anterior neighborhood and unzippering from the posterior one . BetweenMeasurements of in vivo zippering velocities  support Together, the developmental dynamics observed for AIB neurite placement are consistent with relative changes in adhesive forces between the neighborhoods. This suggests that dynamic mechanisms resulting in differential adhesion might govern AIB neurite repositioning by a process similar to affinity-based sorting of cells within homogenous tissues . We showsyg-1 and syg-2, which encode a pair of interacting Ig family cell adhesion molecules (IgCAMs), display significant defects in the placement of the AIB neurite. In wild type animals, we reproducibly observed complete overlap between the AIB distal neurite and neurons in the anterior neighborhood (syg-1(ky652) animals and 60 % of syg-2(ky671) animals  showed regions of AIB detachment from neurons specifically in the anterior neighborhood (syg-1(ky652) and syg-2(ky671) animals that exhibit defects in AIB neurite placement, we found that 20.9 ± 3.9 and 18.6% ± 4.0% (respectively) of the neurite segment in the anterior neighborhood is detached from the neighborhood . We discovered that loss-of-function mutant alleles of hborhood , consisthborhood . In conthborhood . In the hborhood . Our finC. elegans egg-laying circuit  and SYG-2  orthologs also act as multipurpose adhesion molecules in varying conserved developmental contexts (syg-1(ky652) and syg-2 (ky671) loss-of-function alleles did not enhance the AIB distal neurite placement defects as compared to either single mutant (The IgCAMs SYG-1 and SYG-2 are a receptor-ligand pair that has been best characterized in the context of regulation of synaptogenesis in the  circuit . SYG-1 (contexts . In mostcontexts . Consiste mutant .syg-2(ky671) mutants, suggesting that SYG-2 acts cell autonomously in AIB. While expression of wild-type SYG-1 (via a cosmid) rescued AIB neurite placement onto the anterior neighborhood, expression of SYG-1 using an AIB cell-specific promoter did not , we observed robust expression of the hborhood . The SYGhborhood . To undehborhood , using bsyg-1 reporter expression in the nerve ring was primarily restricted to a single band corresponding to the AIB posterior neighborhood  mutants was sufficient to position the AIB distal neurite along these neurons  ingrowth of SYG-1-expressing neurons into the anterior neighborhood and (ii) onset of hborhood . We obsehborhood . To test mutants . We also neurons .syg-1 mutants  mutant background . Unlike wild type and  mutants , animalsl stages . Importackground , consistWe reasoned that if differential adhesion mechanisms were driving zippering of the AIB neurite during development, expression of the SYG-1 ectodomain would be sufficient to drive the ectopic interactions upon misexpression . Indeed,syd-2(ola341) isolated from our screens exhibit synaptic defects, but do not display phenotypes in AIB neurite placement within the anterior neighborhood (syg-1(ky652), RAB-3 signal was specifically and consistently reduced in regions of the AIB distal neurite incapable of repositioning to the anterior neighborhood  mutants. When we fluorescently labeled RIM and AVE in wildtype animals, we observed that the proximal AVE neurite runs along the RIM neurite, consistent with EM studies ) mutants versus 9.1 % in wild type  and 3.99 μm in wild-type animals; We next examined if hborhood  . Detailed cloning information or plasmid maps will be provided upon request. Transgenic strains were generated via microinjection with the construct of interest at 2–100 ng/µL by standard techniques (unc-122p: GFP or unc-122p: RFP were used.We used Gibson Assembly (New England Biolabs) or the Gateway system (Invitrogen) to make plasmids  used forchniques . Co-injesyg-1 transcriptional reporter .For cell-specific labeling and expression in larvae, we used an  for AIB , a ceh-3 and ASE , tdc-1,  for RIM , and an olaIs67) with AIB labeled with cytoplasmic mCherry and AIB presynaptic sites labeled with GFP:RAB-3. Ethyl methanesulfonate (EMS) mutagenesis was performed and animals were screened for defects in placement of the AIB neurite, or presynaptic distribution. We screened for these same phenotypes in our reverse genetic screens as well, where we crossed the marker strain (olaIs67) to characterized mutant alleles. We screened F2 progeny on a Leica DM 5000 B compound microscope with an HCX PL APO 63 x/1.40–0.60 oil objective.We performed a visual forward genetic screen in an integrated wild type transgenic strain (were out-crossed six times to wild type (N2) animals and mapped via single-nucleotide polymorphism (SNP)  (Mutants from forward genetic screens sm (SNP)  and wholsm (SNP) . We anal(ola341) .syg-1(ky652) and syg-2(ky671) exhibited AIB neurite placement defects.We also performed a reverse genetic screen with candidate adhesion molecules expressed in AIB and its primary postsynaptic partner, RIM , for defWe used an UltraView VoX spinning disc confocal microscope with a 60 x CFI Plan Apo VC, NA 1.4, oil objective on a NikonTi-E stand (PerkinElmer) with a Hamamatsu C9100–50 camera. We imaged the following fluorescently tagged fusion proteins, eGFP, GFP, PH:GFP (membrane-tethered), RFP, mTagBFP1, mCherry, mCherry:PH, mScarlet, mScarlet:PH at 405, 488 or 561 nm excitation wavelength. We anesthetized larval stage four animals (unless otherwise mentioned) at room temperature in 10 mM levamisole (Sigma) and mounted them on glass slides for imaging. For We used the Volocity image acquisition software (Improvision by Perkin Elmer) and processed our images using Fiji . Image pFor labeling of neurites in embryos, we used membrane tethered PH:GFP or mScarlet:PH. A subtractive labeling strategy was employed for AIB embryo labeling . Brieflyhttp://www.cytoshow.org/ as described   and a coescribed .We developed a triple-view microscope that can sequentially capture three specimen views, each acquired using line-scanning confocal microscopy . Multivihttp://promoters.wormguides.org. Our integrated imaging and lineaging approaches enabled us to identify a promoter region of inx-19 which is expressed in the RIM neurons prior to RIM neurite outgrowth (~370 m.p.f.) and in additional neurons in later embryonic stages. The inx-19p was one of the promoters used for embryonic ablation of the RIM neurons (described in the next section).Cell lineaging was performed using StarryNite/AceTree . Light snphp-4p and mgl-1bp). 4/4 neuron classes that were identified to have nphp4p expression are in the AIB posterior neighborhood  and 2/3 neuron classes that were identified to have mgl-1bp expression are in the AIB posterior neighborhood  (http://promoters.wormguides.org). We used these promoters to drive ectopic expression of a syg-1 cDNA specifically in the posterior neighborhood.Our integrated imaging and lineaging approach also enabled us to identify two promoters with expression primarily in neurons located at the AIB posterior neighborhood . The cell identities obtained for the left and right sides of the nerve ring were consistent.We also used this imaging and lineaging approach to identify SYG-1 expressing neurons in the anterior and posterior neighborhoods . We deteinx-19p (termed ablation strategy 1), and another set of ablation strains with co-expression of the p12 subunit expressed under inx-19p and p17 under tdc-1p (termed ablation strategy 2) .The RIM neurons were ablated using a split-caspase ablation system . We geneategy 2) . L3 larvhttps://imagej.net/Fiji#Downloads) and saved as object files (.obj), or by using the 3D viewer in CytoSHOW.From available EM datasets  we rendenm) and identified for overlap by comparing with neighboring undilated ROIs from the same EM slice. A collection of 289,012 regions of overlap were recorded as new ROIs, each bearing the color code of the dilated ROI and labeled with both cell-names from the pair of the overlapped ROIs. These ‘contact patch’ ROIs were then grouped by cell-pair-name and rendered via a marching cubes algorithm to yield 3D isosurfaces saved in.obj files. Each of the 8852 rendered.obj files represents all patches of close adjacency between a given pair of neurons, color-coded and labeled by cell-pair name. Selected.obj files were co-displayed in a CytoSHOW3D viewer window to produce views presented in Figure 1, Figure 1—figure supplement 1 and Figure 1—figure supplement 2.To generate 3D mappings of inter-neurite membrane contact, the entire collection of 76,046 segmented neuron membrane boundaries from the JSH TEM datasets  were impC. elegans in http://openworm.org - 3D Model by Christian Grove, WormBase, CalTech).The schematic representations of larval We performed cosine similarity analysis  on AIB cwhere A and B are the two vectors under consideration with the symbol ‘‘ denoting the i-th entry of each vector. The similarity values were plotted as a heat map for AIBL and AIBR using Prism. For the datasets L1_0 hr, L1_5 hr, L1_8 hr, L2_23 hr, L3_27 hr, L4_JSH and Adult_N2U, only the neuron-neuron contacts in the EM sections corresponding to the nerve ring were used (as opposed to the whole connectome).We analyzed betweenness centrality for two of the available connectomes of different developmental stages (L1 and adult) . By treaHere  Toolbox  of MATLA Toolbox  correspo Toolbox . We madeSince we observed that the proximal and distal neurites of AIBL and AIBR completely align and overlap  in confoThe penetrance of defects in AIB neurite placement in the anterior neighborhood in mutant (or ablation) strains was determined by visualizing the AIB neurite and scoring animals with normal or defective anterior neighborhood placement under the Leica compound microscope described. Animals in which the entire distal neurite was placed at a uniform distance from the proximal neurite, for both AIBL and AIBR, were scored as having normal AIB distal neurite placement.The penetrance of the gain-of-function effects in ectopic SYG-1 expression strains was determined by scoring the percentage of animals showing ectopic AIB distal neurite placement in the posterior neighborhood. Animals with part (or whole) of the AIB distal neurite overlapping with the posterior neighborhood were considered as having ectopic AIB placement.Minimum perpendicular distances between neurites  were mead) x 100/ total length (Lt) .The percent detachment for defasciculated neurites (AIB or AVE and RIM) is calculated by the formula % detachment = detached length (Lt) and only the portion of the distal neurite positioned in the posterior neighborhood (Lp) are measured using Fiji. (Lp/Lt)x100 provides the percentage of the distal neurite placed in the posterior neighborhood.Freehand line selections of the entire distal neurite (Lsyg-1 reporter expression in embryos (syg-1p) = mean anterior neighborhood intensity (Ia)/mean posterior neighborhood intensity (Ip). These measurements were done in transgenic embryos co-expressing the AIB reporter and the syg-1 transcriptional reporter. For calculation of Ip, a freehand line selection was made  enrichment values .Relative (anterior) enrichment of  embryos  is calcut values . These vThe dorsal midline shift (chiasm) lengths of AIB and AVE were calculated by making 3D maximum intensity projections of confocal z-stacks and orienting the neuron pair to a dorsal-ventral view. A straight line selection is made along the posterior-anterior shift of each neuron, and each arm of the ‘X’ of the chiasm was measured (using Fiji).The length of the distal neurite of AIB was measured by drawing a freehand line along the neurite segment occupying the distal neighborhood (including the chiasm) in maximum intensity projections of confocal image stacks where the worms are oriented on their side .C. elegans larvae and image processing.’.The positions of the AIB neurite in the anterior and posterior neighborhoods in nphp-4 promoter (see Results and http://promoters.wormguides.org). All measurements are performed using CytoSHOW.The angle of exit (α) of the developing AIB distal neurite is measured as the angle between straight line tangents drawn along the separating distal segment of AIBL and the proximal neurite of AIBR and vice versa. These measurements are performed on deconvolved maximum intensity projections of diSPIM images where the neurons are oriented in an axial view. The angle of ventral turn of the nerve ring (β) is measured as the angle between straight line tangents drawn along segments of the nerve ring on either side of the ventral bend of the nerve ring in the posterior neighborhood see . β is meTime-lapse imaging of presynaptic proteins RAB-3, CLA-1, and SYD-2 in AIB in embryos was performed using diSPIM . To visuThe line intensity plot in cnd-1 (syg-1(ky652) mutant animals. Measurements were done on confocal image stacks where the worms are oriented on their side . On each side of the worm, a straight line selection along the anterior-posterior axis from one edge of the labeled nerve ring to the other was defined as the nerve ring width.The nerve ring was visualized using a 5.6 kb promoter of cnd-1  driving z). We then calculated the x-y distance between the segmented regions of the AIBL shift in the topmost and bottommost z-slice(dx-y). We calculate the length of the shift in 3D (l) using the formulaFrom a segmented EM dataset of the L4 larva JSH , we calcThe same measurements were repeated for the length of the dorsal midline shift of AIBR.Statistical analyses were conducted with PRISM seven software. For each case, the chosen statistical test is described in the figure legend and ‘n’ values are reported. Briefly, for continuous data, comparisons between two groups were determined by unpaired two-tailed t-test and comparisons within multiple groups were performed by ordinary one-way ANOVA. Error bars were reported as standard error of the mean (SEM). For categorical data, groups were compared with two-sided Fisher’s exact test. The range of p-values for significant differences are reported in the figure legend. The Cohen’s d statistic was determined for comparisons between continuous datasets with statistically significant differences, to obtain estimates of effect sizes. Your work provides novel and interesting insights into circuit formation, demonstrating how synaptic specificity is controlled at least in part by different cell adhesion during neurite placement. The revisions of your paper have addressed the points raised by the reviewers and we are glad to see that those revisions have further strengthened the conclusion of this paper. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Piali Sengupta as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Kang Shen (Reviewer #3).Thank you for submitting your article "Differential adhesion regulates neurite placement via a retrograde zippering mechanism" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:After discussion among the three reviewers, the only experiment that two of the reviewers considered important to add is an analysis of an active zone marker (e.g. syd-2) to strengthen the conclusion that synapse formation only occurs after zippering. Currently, the way you discuss the zippering event with synapse formation is vague. If a marker like SYD-2 shows the same time course as RAB-3, then, a strong statement would be made that zippering happens before synapse formation and functions as a separate specificity mechanism. The reviewers think this is not a hard experiment to do and it will also validate the time course of GFP::RAB-3.The other experimental request voiced by reviewer #1, regarding an improvement of the syg-1/2 expression analysis was, after discussion, deemed to be considered essential. Further discussion in the text that may assuage the concerns raised by reviewer #1 may be warranted.All other requested revisions are editorial/textual/organizational in nature, but we ask you to nevertheless take them very seriously since several of them deal with important quantification issues.Reviewer #1:C. elegans nerve ring. The authors show that neighborhood placement of the interneuron AIB is established during embryogenesis and is maintained throughout development. AIB is one of the few C. elegans neurons that are divided into distinct pre- and post-synaptic regions, and its axons curiously occupy two physically separated neighborhoods or layers. How this occurs is not known. This study uses time-lapse imaging to show that unlike canonical axon tip outgrowth mediating fasciculation in a target region, AIB's axon occupies two neighborhoods by first growing completely into one, and then gradually unzippering from the first, switching, and zippering onto the second neighborhood. Importantly, axon outgrowth and neighborhood choice are continuously visualized during embryogenesis, an impressive experiment typically constrained by lack of cell-specific reporters during early development as well as the struggle of imaging embryos.In their manuscript, Sengupta et al. describe a developmental mechanism that positions a single neuron across multiple layers in the hierarchical The authors posit that zippering is mediated by temporally regulated differential adhesive forces between AIB's neighboring pre- and post-synaptic neurons. How this differs from differential adhesion in classic fasciculating neurons is described but could be made much clearer. They proceed to identify the immunoglobulin syg-1/syg-2 receptor-ligand pair to be necessary and sufficient for AIB's axon switch; in syg-1/syg-2 mutants, AIB is not able to position itself in the second neighborhood and remains fasciculated with the first one, suggesting that adhesive forces are dampened in syg-1/syg-2 mutants. Lastly, the authors show that pre-synapse assembly follows zippering, linking AIB axon placement with synaptogenesis, and that this is also compromised in syg mutants.C. elegans nervous system establishment. Although the overall manuscript and data are well-presented, we think the mechanism of retrograde zippering could be better described. Also, syg-1/syg-2 expression needs to be delineated to support the notion of differential adhesion between neighborhoods.The pipeline used to study axon outgrowth at a single-cell level in the embryo at relevant time points is commendable and will be useful to people studying Major revisions:1. The process of zippering can be described in more detail and contrasted with canonical axon tip outgrowth and selective fasciculation, especially in the context of differential adhesion and biophysical forces mediating cell adhesion. Drawing on more literature that describes zippering in other contexts might be helpful.2. Figure 5: syg-1 expression changes and enrichment in the anterior neighborhood warrant better characterization. The authors write that "syg-1 transcriptional reporter shows banded expression in ~20 neurons present in the AIB posterior and anterior neighborhoods, with specific enrichment in anterior neighborhood" but identities of these neurons are not characterized using the transcriptional reporter. This is relevant because if there are more neurons in one neighborhood compared to the other, then you would naturally see more expression (given the method of quantification) but it wouldn't necessarily be "enrichment" since more neurons are growing into that neighborhood at that point in development and those additional neurons might not be synapsing with AIB. Or they might be the only neurons synapsing with and thus attracting AIB, in which case differential levels of syg-1 are not as important as presence of it. Understandably, quantifying embryonic expression at the single-cell level is not trivial in the absence of cell specific reporters in the background, but could the authors characterize expression at L1 (since the enrichment and AIB axon placement are maintained)? Could the authors use an endogenous nuclear-localized reporter for syg-1 to aid quantification and glean cell identities, given candidates from Packer et al. data and their own experiments?3. Related to point 1, enrichment of translational SYG-1 reporter in the anterior neighborhood is not obvious.4. [line 365] The authors state that "SYG-1 expression in RIM neurons contributes to AIB neurite placement" but Packer et al. data oddly does not show a temporally dynamic enrichment in RIM (supp table 1) at the time AIB switches neighborhoods. Could it be that AIB only switches to the anterior neighborhood once the target neuron RIM grows into it? If so, it's unclear why this would be termed enrichment. The authors need a more robust readout for temporal syg-1 expression in RIM.Reviewer #2:A large amount of data is presented in this paper. The experiments are carefully documented and support the conclusions. Of particular importance is the live imaging of the outgrowth of the AIB neurite in the embryo. This is challenging and required the development of a new marker for labelling and the adaptation of a new type of microscope. This enabled the initial and surprising observation that part of the neurite relocates after outgrowth. I'm not sure that the mathematical modeling adds much. The main conclusion is that the modeling is consistent with a "net increase of adhesive forces in the anterior neighbourhood", which is to be expected. The authors then try to identify the relevant adhesion molecules and find that a pair of IgCAMs (syg-1 and syg-2), which are known to act as receptor-ligand pair, are involved. A series of experiments establishes that syg-2 act in the AIB neurons, whereas syg-1 does not. The neurite positioning defects in syg-1 and syg-2 mutants are partially penetrant, suggesting that other adhesion molecules must be involved. While a large percentage of mutant animals show defects, the defects within an individual animal are surprisingly low with only 21.5% +/- 4% of the neurite detached. This would suggest that syg-1/syg-2 aren't even the major adhesion molecules involved here. In further studies, where the authors ablate the RIM neurons (which express syg-1), the authors use a different measure to quantify the defects . This makes it difficult to compare the results to those of the syg-1 mutants. For the ectopic expression experiments with syg-1 the authors only report the percentage of animal with defects and not the extent of the defects .Overall, this is a very detailed study describing an important novel mechanism for neurite positioning within an nerve bundle.Reviewer #3:C. elegans embryos which are very hard to do. The surprising finding here is that neurite position is not sole dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shift direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2. Overall, I think this is a strong candidate for eLife.This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript included some technically difficult experiments of imaging neurite outgrowth in My main point is about the relationship between synapse formation and neurite zippering. In my opinion, this is an interesting point because it would tell us if the zippering behavior is a consequence of synapse formation or it is a distinct specificity step before synapse formation. From the time course that was described in the paper, it seems that the accumulation of RAB-3 only starts after the zippering has completed. I would suggest the authors to examine at least another synaptic marker like SNB-1 or SYD-2. We have created cell specific endogenous labeling of several active zone markers that can be used for these experiments. If the results hold, then, I think the authors should make it clear in the text that the zippering takes place before synapse formation and serves as a distinct step in achieving the neighborhood specificity.Examine the localization of at least one active zone proteins and compare the time course of synapse formation to the zippering of AIB neurite. Reviewer #1:C. elegans nerve ring. The authors show that neighborhood placement of the interneuron AIB is established during embryogenesis and is maintained throughout development. AIB is one of the few C. elegans neurons that are divided into distinct pre- and post-synaptic regions, and its axons curiously occupy two physically separated neighborhoods or layers. How this occurs is not known. This study uses time-lapse imaging to show that unlike canonical axon tip outgrowth mediating fasciculation in a target region, AIB's axon occupies two neighborhoods by first growing completely into one, and then gradually unzippering from the first, switching, and zippering onto the second neighborhood. Importantly, axon outgrowth and neighborhood choice are continuously visualized during embryogenesis, an impressive experiment typically constrained by lack of cell-specific reporters during early development as well as the struggle of imaging embryos.In their manuscript, Sengupta et al. describe a developmental mechanism that positions a single neuron across multiple layers in the hierarchical The authors posit that zippering is mediated by temporally regulated differential adhesive forces between AIB's neighboring pre- and post-synaptic neurons. How this differs from differential adhesion in classic fasciculating neurons is described but could be made much clearer. They proceed to identify the immunoglobulin syg-1/syg-2 receptor-ligand pair to be necessary and sufficient for AIB's axon switch; in syg-1/syg-2 mutants, AIB is not able to position itself in the second neighborhood and remains fasciculated with the first one, suggesting that adhesive forces are dampened in syg-1/syg-2 mutants. Lastly, the authors show that pre-synapse assembly follows zippering, linking AIB axon placement with synaptogenesis, and that this is also compromised in syg mutants.C. elegans nervous system establishment. Although the overall manuscript and data are well-presented, we think the mechanism of retrograde zippering could be better described. Also, syg-1/syg-2 expression needs to be delineated to support the notion of differential adhesion between neighborhoods.The pipeline used to study axon outgrowth at a single-cell level in the embryo at relevant time points is commendable and will be useful to people studying syg-1 expression .We have further clarified the novelty of the zippering mechanism, contrasting it with tip-directed outgrowth. We have also performed a thorough analysis of Major revisions:1. The process of zippering can be described in more detail and contrasted with canonical axon tip outgrowth and selective fasciculation, especially in the context of differential adhesion and biophysical forces mediating cell adhesion. Drawing on more literature that describes zippering in other contexts might be helpful.We have added new textual clarification on how differential adhesion gives rise to the observed zippering mechanism, and distinguished this mechanism from canonical tip-directed outgrowth . While retrograde zippering had been described for tissue culture cells, it had not been documented in vivo, and its importance, if any, remained unknown. We now better explain in the text that our study is, to our knowledge, the first in vivo demonstration of the role of retrograde zippering in neurite placement during development.2. Figure 5: syg-1 expression changes and enrichment in the anterior neighborhood warrant better characterization. The authors write that "syg-1 transcriptional reporter shows banded expression in ~20 neurons present in the AIB posterior and anterior neighborhoods, with specific enrichment in anterior neighborhood" but identities of these neurons are not characterized using the transcriptional reporter. This is relevant because if there are more neurons in one neighborhood compared to the other, then you would naturally see more expression (given the method of quantification) but it wouldn't necessarily be "enrichment" since more neurons are growing into that neighborhood at that point in development and those additional neurons might not be synapsing with AIB. Or they might be the only neurons synapsing with and thus attracting AIB, in which case differential levels of syg-1 are not as important as presence of it. Understandably, quantifying embryonic expression at the single-cell level is not trivial in the absence of cell specific reporters in the background, but could the authors characterize expression at L1 (since the enrichment and AIB axon placement are maintained)? Could the authors use an endogenous nuclear-localized reporter for syg-1 to aid quantification and glean cell identities, given candidates from Packer et al. data and their own experiments?syg-1 expression levels that regulate AIB neurite transition between neighborhoods by zippering. We present these findings in new Figure 5—figure supplement 2 and discuss them in lines 354-361 and 1536-1565.We identify the SYG-1 expressing neurons in embryos by performing single-cell lineage analysis as described  and identify sixteen SYG-1 expressing neurons in the nerve ring: six in the anterior neighborhood, and ten in the posterior neighborhood . Our findings, which are consistent with transcriptomic datasets , extends our understanding of SYG-1 nerve ring expression during development, and are consistent with our observations on dynamic 3. Related to point 1, enrichment of translational SYG-1 reporter in the anterior neighborhood is not obvious.We have revised Figure 5, including a timepoint before zippering  and one during zippering , to more clearly represent the increase of SYG-1 in the anterior neighborhood.4. [line 365] The authors state that "SYG-1 expression in RIM neurons contributes to AIB neurite placement" but Packer et al. data oddly does not show a temporally dynamic enrichment in RIM (supp table 1) at the time AIB switches neighborhoods. Could it be that AIB only switches to the anterior neighborhood once the target neuron RIM grows into it? If so, it's unclear why this would be termed enrichment. The authors need a more robust readout for temporal syg-1 expression in RIM.We use the term “SYG-1 enrichment” to refer to increase in SYG-1 expression levels in the entire anterior neighborhood. These increases can be caused by (i) ingrowth of SYG-1-expressing neurons into the neighborhood and (ii) increase in SYG-1 levels in neurons already in the neighborhood. The RIM neurons grow into the anterior neighborhood, contributing to the local increase of overall SYG-1 levels in this neighborhood. We have clarified this in the text (lines 377-378) and also now provide a more extensive characterization of the SYG-1 expressing neurons in embryos (as described in Comment 2 above).Reviewer #2:A large amount of data is presented in this paper. The experiments are carefully documented and support the conclusions. Of particular importance is the live imaging of the outgrowth of the AIB neurite in the embryo. This is challenging and required the development of a new marker for labelling and the adaptation of a new type of microscope. This enabled the initial and surprising observation that part of the neurite relocates after outgrowth. I'm not sure that the mathematical modeling adds much. The main conclusion is that the modeling is consistent with a "net increase of adhesive forces in the anterior neighbourhood", which is to be expected. The authors then try to identify the relevant adhesion molecules and find that a pair of IgCAMs (syg-1 and syg-2), which are known to act as receptor-ligand pair, are involved. A series of experiments establishes that syg-2 act in the AIB neurons, whereas syg-1 does not. The neurite positioning defects in syg-1 and syg-2 mutants are partially penetrant, suggesting that other adhesion molecules must be involved. While a large percentage of mutant animals show defects, the defects within an individual animal are surprisingly low with only 21.5% +/- 4% of the neurite detached. This would suggest that syg-1/syg-2 aren't even the major adhesion molecules involved here. In further studies, where the authors ablate the RIM neurons (which express syg-1), the authors use a different measure to quantify the defects . This makes it difficult to compare the results to those of the syg-1 mutants. For the ectopic expression experiments with syg-1 the authors only report the percentage of animal with defects and not the extent of the defects .Overall, this is a very detailed study describing an important novel mechanism for neurite positioning within an nerve bundle.syg-1(ky652) and syg-2(ky671) mutants, allowing direct comparisons between the phenotypes. We have also added a measure for the percentage of the distal neurite that is mispositioned in the ectopic expression experiments . We do not claim that SYG-1/SYG-2 are the only adhesion molecules involved in AIB neurite placement, but that they are required for complete and proper placement of the neurite. We clarify this in the text.We have added in this revised version additional quantifications, including ‘the minimal distance between neurite segments’ measure (the one used for the RIM ablation experiments) in Figure 4—figure supplement 1 for the placement defects in Reviewer #3:C. elegans embryos which are very hard to do. The surprising finding here is that neurite position is not sole dependent on its growth cone navigation. In the case of the AIB neuron, the growth cone is anchored after it reaches its destination point and then a segment of the neurite shift direction towards its final position through a zippering action. They also show that this shift in position is driven by adhesion molecules SYG-1 and SYG-2. Overall, I think this is a strong candidate for eLife.This is a very interesting manuscript describing the changes of neurite position in a complex neuropil during development. The experimental system is well chosen because AIB's function within the circuit requires its neurite to be in two different neuropil "neighborhoods". The manuscript included some technically difficult experiments of imaging neurite outgrowth in My main point is about the relationship between synapse formation and neurite zippering. In my opinion, this is an interesting point because it would tell us if the zippering behavior is a consequence of synapse formation or it is a distinct specificity step before synapse formation. From the time course that was described in the paper, it seems that the accumulation of RAB-3 only starts after the zippering has completed. I would suggest the authors to examine at least another synaptic marker like SNB-1 or SYD-2. We have created cell specific endogenous labeling of several active zone markers that can be used for these experiments. If the results hold, then, I think the authors should make it clear in the text that the zippering takes place before synapse formation and serves as a distinct step in achieving the neighborhood specificity.inx-1 and unc-42 promoters for cell-specific labeling of these active zone proteins in AIB and injected them into the strains. Although we observed cell-specific synaptic signal in larvae with both inx-1p and unc-42p-driven FLP, we were not able to observe signal during embryogenesis, probably due to cell-specific synaptic protein expression levels being low.We thank the reviewer for generously sending us strains for cell-specific endogenous active zone protein labeling  using the SapTrap method . We made constructs expressing FLP recombinase downstream of the syd-2(ola341) allele identified in our screen, we find that although synapses are mislocalized, AIB neurite placement is unaffected, consistent with the idea that synapse formation is not upstream of zippering-mediated placement .Therefore and to address the reviewer’s question about the temporal order of zippering and synapse formation, we have cell-specifically expressed two active zone proteins in AIB (CLA-1 and SYD-2) and measured their intensities over time in AIB in embryos . We find that similar to RAB-3, synaptic signal is not visible until after the end of zippering, and progressively increases over time following zippering. These observations suggest that synapses do not initiate retrograde zippering. We added the time-course of active zone protein localization in AIB in the context of the time course of retrograde zippering . Consistent with these observations, in a We acknowledge, however, that our studies are limited by detection of the synaptic proteins, and that, while it does not appear that synaptogenesis leads to zippering, a cooperative and synergistic relationship might exist between the process of zippering and synaptogenesis to hold the neurite position in place. We have added text to better discuss this relationship between zippering and synaptogenesis in light of these findings.Examine the localization of at least one active zone proteins and compare the time course of synapse formation to the zippering of AIB neurite.We have included the time course of localization for SYD-2 and CLA-1 and shown that they occur concurrently with RAB-3 localization  and after retrograde zippering of the AIB neurite , supporting our conclusions. We now better explain the model of how we envision the interaction between synaptogenesis and neurite placement in lines 421-440, 545-559.ReferencesCaenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America.Bao, Z., Murray, J. I., Boyle, T., Ooi, S. L., Sandel, M. J. and Waterston, R. H. 2006. Automated cell lineage tracing in Nature, 595, E35.Mcdonald, N. A., Fetter, R. D. and Shen, K. 2021. Author Correction: Assembly of synaptic active zones requires phase separation of scaffold molecules. C. elegans embryogenesis at single-cell resolution. Science, 365.Packer, J. S., Zhu, Q., Huynh, C., Sivaramakrishnan, P., Preston, E., Dueck, H., Stefanik, D., Tan, K., Trapnell, C., Kim, J., Waterston, R. H. and Murray, J. I. 2019. A lineage-resolved molecular atlas of Caenorhabditis elegans. Genetics, 202, 1277-88.Schwartz, M. L. and Jorgensen, E. M. 2016. SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 314, 1-340.White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. 1986. The structure of the nervous system of the nematode


Oral HIV self-testing (HIVST) may be useful for increasing testing in persons at elevated risk of acquiring HIV.We conducted a pilot study to evaluate the feasibility (defined by uptake) of HIVST among FSW in Gaborone, Botswana. FSW age 18 years and above were recruited through a non-governmental organization serving FSW. FSW with unknown or negative HIV status at screening performed HIVST in the study clinic following brief training. FSW testing HIV-negative were each given two test kits to take home: one kit to perform unassisted HIVST and another to share with others. Feasibility (use) of HIVST (and sharing of test kits with others) was assessed in these women at a study visit four months later.Two hundred FSW were screened. Their average age was 34 years (range 18–59), and 115 (58%) were HIV-positive. Eighty-five (42%) tested HIV-negative at entry and were eligible to take part in the HIVST pilot study. All 85 (100%) agreed to take home HIVST kits. Sixty-nine (81%) of these 85 participants had a follow-up visit, 56 (81%) of whom reported performing HIVST at a mean of three and half months after the initial visit. All 56 participants who performed HIVST reported negative HIVST results. Fifty (73%) of the 69 participants who took HIVST kits home shared them with others. Of the 50 women sharing HIVST kits, 25 (50%) shared with their non-client partners, 15 with a family member, 8 with friends, and 3 with a client. One participant did not test herself but shared both her test kits. Most participants 53/56 (95%) found oral HIVST very easy to use whilst 3/56 (5%) felt it was fairly easy.Oral HIVST is feasible among FSW in Gaborone, Botswana. The majority of FSW used the HIVST kits themselves and also shared extra HIVST kits with other individuals. HIV infection remains a major public health challenge, particularly in the sub-Saharan region , 2. HeteHIV self-testing has not only been shown to be safe but to also increase frequency of testing among some key populations including FSW , 26. In Individual HIV self-testing, coupled with secondary distribution of HIV self-test kits, have been shown to be effective HIV prevention strategies in some hard to reach populations in sub-Saharan Africa –34. StudAgainst this background we aimed to evaluate the feasibility (uptake) of oral HIV self-testing among FSW (and sharing of HIVST kits with others) in Gaborone, Botswana.We conducted a pilot study among FSW in Gaborone Botswana between June 2019 and February 2020 to evaluate the feasibility of oral HIV self-testing. Feasibility was defined as uptake of the intervention (described in detail in “Outcomes” section below) , 41. We The “Ikitse Study “(“know yourself” in Setswana) was approved by the following institutional review boards: the Health Research and Development Committee of the Botswana Ministry of Health and Wellness (HRDC#00842), and the Office of Human Research Administration at the Harvard T.H. Chan School of Public Health (IRB18-1351). All participants provided written informed consent.To be eligible for the study, participants needed to be 18 years-old or older and identify as a FSW. Specifically, women needed to confirm receipt of money or goods in exchange for sexual services within the past three months, and consciously define those activities as income-generating even if they did not consider sex work as their primary occupation . At the Participants were recruited by peer FSW outreach workers affiliated with the Nkaikela Youth Group, a non-governmental organization that provides services to FSW in and around Gaborone area. Women who received services from the Nkaikela Youth Group contacted the study team to self-refer for this pilot study, and referred other FSW to participate . Participants were then scheduled to come for a screening and entry visit.First, history and documentation of prior HIV testing (and test result) were obtained. Women who were not already documented to be HIV-positive were offered HIV counselling and testing using the OraQuick HIV self-testing kit, which has sensitivity of 90–98% and specificity of 98–100% , 44. TraWomen who tested HIV-positive were counselled and referred for prompt confirmatory HIV testing and ART initiation if not already on treatment. Women with HIV also completed a brief one-time questionnaire about HIV and ART history and did not have subsequent study procedures.Women whose entry visit oral HIV self-test was negative were offered two self-test kits to take home; one for them to test themselves, and a second kit to either share with another individual of their choosing or to use for another self-test on themselves. Women who declined taking the HIV self-testing kits home were to be invited for an in-depth interview to provide insights into reasons for declining. Women whose HIV self-test was negative were also counseled about and referred for tenofovir/emtricitabine oral pre-exposure prophylaxis (PrEP) to prevent HIV acquisition, which is available at no cost for key populations in Botswana.Participants who accepted the oral HIV self-test kits were provided with written step-by-step instructions from the manufacturer for using the HIV self-test, with diagrams and in participants’ preferred language (English or Setswana). Participants were also provided with a care card (one for each kit), on which the participant was asked to document the date and result of the HIV self-test. The care card also included guidance for next steps based on the self-test result, including contact information for rapid access to counselling and treatment services for participants who had a positive result (or to contact study staff if they had questions about how to perform the self-testing).Women were counselled to test themselves using the HIV self-test kit approximately three months after the initial visit. They were given the option to decide whether or not study staff can send them a reminder text/SMS message to use the kit after three months. Women were counselled that they could use the second test kit to test themselves at any time that they wish—or, if they preferred, to test another adult who wished to and agreed to be HIV-tested, or to give the test kit to another individual with the written instructions for use. If the participant tested another individual, she was asked to give the individual a care card.Participants were asked to bring their care card to the follow-up appointment, in order to document whether or not they performed the self-test. Research staff emphasized to participants that they would not be judged negatively if they did not use the self-test kit, and highlighted that what was important was to understand whether the self-test was truly acceptable and feasible . During the follow-up visit, participants were asked (using a structured questionnaire) about whether and when they performed the HIV self-test, the test result, their perceptions of self-testing, whether or not they were able to offer to test someone else with the extra self-test (and whom), and the advantages and the challenges/barriers associated with self-testing. Participants were reimbursed approximately $7 as compensation for transport and time at each visit, consistent with the Institutional Review Board approval.The primary outcome of interest was feasibility of HIV self-testing, defined as the proportion of women who used an HIV self-test during follow-up, among all women who were offered HIV self-test kits to take home. Secondary outcomes included the number of participants who passed on the extra self-test kit to someone else, and participant impressions of self-testing.Statistical analysis was performed using STATA(version 16.0). FSW demographics and other characteristics at entry visit were summarized using frequencies, proportions for categorical variables; and means and ranges for continuous variables. Univariate analyses were conducted to evaluate the association between socio-demographic factors and reported use of HIV self-test kits.Between June 2019 and September 2019, 200 FSW were recruited from Gaborone and surrounding areas. Of these 200 women, 199/200 (99.5%) had previously tested for HIV, 110 (55%) of whom had known HIV positive status at entry. Of the 90 women who underwent HIV self-testing at entry, an additional 5 tested positive and were referred for care and treatment. Hence, the overall prevalence of HIV was 58%. One hundred and seven (93%) of the 115 women living with HIV were taking ART (thus 107 [97%] of the 110 women who knew their positive status were taking ART).Eighty-five (42%) of the 200 women recruited to the study were HIV-negative and were eligible to take part in the pilot study of HIV self-testing. All 90 participants who were not already documented to be living with HIV accepted in-clinic HIV testing. They all performed assisted HIVST to the satisfaction of the study staff. All (100%) 85 women who tested HIV-negative accepted HIV self-testing kits to take home. Of these, 69/85 (81%) had follow up visits conducted whilst the remaining 16 women were lost to follow up despite multiple contact attempts. Fifty six (81%) of the 69 participants who followed up reported performing the HIV self-test, at a mean of 3.5 months after the initial visit . All 56 participants who self-tested and followed up reported that the HIV self-test result was negative. None of the women used both test kits to test themselves. Of the 13 women who had a follow-up visit and who did not perform HIV self-testing, 6 either misplaced/lost the self-test kit, 3 were afraid to test, 3 changed their minds and no longer wanted to test, and one opted to test two people other than herself.Of the 56 participants who had a follow up visit and self-tested; 20 tested alone; 19 tested in the presence of a family member, 12 tested in the presence of their non-client/primary partner, 3 tested in presence of a friend, and 2 tested with someone else. Forty nine participants HIV self-tested in their homes. None of the participants reached out to study staff for assistance with testing or interpreting the test result. Most participants 53/56 (95%) found performing oral HIV self-testing to be very easy whilst 3/56 (5%) felt it was fairly easy. None of the participants thought it was fairly difficult or difficult. Fifty (74%) of 69 participants shared at least one of the two oral HIV self-test kits with other individuals: 25 women shared the test kit with their non-client partners, 15 with a family member, and 8 with friends; 3 tested a client. One participant opted not to test herself and shared both her test kits, noting that she felt they needed them more than she did.HIV testing is an important HIV prevention strategy, particularly for populations that are most vulnerable and hard to reach, We evaluated the feasibility of HIV self-testing among female sex workers in Gaborone Botswana. All HIV-negative women accepted self-test kits to take home. At least two thirds of women who were given oral HIV self-test kits to take home reported using them; uptake may have been higher, given that some of the ~20% of participants who were lost to follow-up after taking home HIVST kits may also have self-tested. Secondary distribution of the self-test kits by FSW to family members, friends and clients was also shown to be feasible and acceptable, with three quarters of women sharing a test kit with another individual. Our results suggest that HIV-self-testing by FSW (and dissemination of HIV self-test kits by FSW to others) is acceptable and feasible, and is a viable component of programs in sub-Saharan Africa that are scaling up HIV self-testing , 45, 46.Our participants were generally aware of their HIV risk, and over 70% had some secondary education. Despite this, only 4% of the participants had ever taken HIV PrEP, which was rolled out in Botswana starting in August 2018 for persons at increased risk of HIV infection, lower than uptake in other countries in the region , 48. TheParticipants reported that oral HIV self-testing was very easy. No participants required any assistance with testing, consistent with findings from other research studies , 49. ParOur findings suggest that secondary distribution of test kits by female sex workers is also feasible. Half of the participants who passed on a test kit did so to a non-client sexual partner, but distribution to clients was low, in contrast to other studies in the region , 31. RepWe found high HIV prevalence (58%), particularly for a group of women who presented with interest in an HIV self-testing study. However, our sample was not random nor geographically dispersed, and HIV prevalence data should be interpreted with caution. ART coverage was very high .Limitations of our study included small sample size, sampling in one region of the country, and non-random sampling  by FSW appears to be a viable HIV testing approach in Botswana.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(XLSX)Click here for additional data file.


The sea covers more than 70% of Earth’s surface and contains more than 300,000 organisms with huge biodiversity. These organisms represent an enormous tank of substances whose chemical structures are the result of the enormous ecological pressure for survival. Given their high chemical diversity, biological activity, biochemical specificity and other crucial molecular properties, marine natural products have always played a crucial role in the search for and discovery of novel bioactive molecules potentially useful for pharmacological applications. In fact the development of new “lead” compounds from the sea has always been—and is one of the main purposes of drug discovery .The study and advanced progress of these products cannot be separated from the development of chemical synthesis and synthetic strategies aimed at the preparation and optimization of these substances and/or analogs, opening the way to new classes of biologically active compounds with pharmacological potential.This Special Issue, comprising eight articles and one review, describes the synthetic methodologies and biological activity of different classes of bioactive marine metabolites and analogs crucial to favor pharmacological applications of these molecules.Esposito et al. , with thMazzotta et al. developed a synthetic strategy for the preparation of different new marine-derived labdane diterpenes analogs, characterized by the homodrimane backbone bearing flexible tails with various chemical properties . They st3•Et2O as activator and water as initiator. Aureol and 5-epi-aureol obtained by this strategy are key intermediates opening the way for the synthesis of a large number of natural and synthetic tetracyclic meroterpenoids with potential antitumor and antiviral activity.Martinez et al. showed a concise strategy for the preparation of sponge derived tetracyclic meroterpenoids characterized by a sesquiterpene skeleton linked to a phenolic or quinone part . Aureol,O-α-D-Glucopyranosyl Tubercidin, a disaccharide 7-deazapurine nucleoside characterized by fungicidal activity [O-glycosylation of 7-deazapurine nucleoside derivative with 2,3,4,6-tetra-O-benzyl-glucopyranosyl trichloroacetimidate.Ouyang et al. described the first eight steps of the total synthesis of 5′-activity . The chel-Cysteine or Glycine moieties was performed, leading to derivatives with totally different biological activity. In particular the S-Me l-Cysteine-modified analog displayed enhanced inhibition of all the tested HDACs. Furthermore, a molecular modeling analysis provided a rational explanation and structural evidence for the enhanced inhibitory activity. This new finding will aid the design of novel, potent HDAC inhibitors.Zhang et al. showed how the substitution on the defined position on the chemical structure of bioactive molecules analogs could be a determinant for biological activity . In partEldehna et al. designed and synthetized different anticancer marine inspired indoles and bis-indoles, as Topsentin and Nortopsentin analogs . This syO-alkylglycerols containing different functional groups  on the alkyl chain and antimicrobial activity was evaluated for all compounds [O-alkylglycerols can be further explored as a new source of drugs.Pemha et al., starting from recinoleic acid, synthetized a series of 1-ompounds . The hydAplidium conicum and named aplidinone A [Imperatore et al. reported the chemical synthesis of two prenyl-quinones and their corresponding dioxothiazine fused quinones . The syndinone A ,15,16. TVessella et al. in their review have collected different and representative reports regarding the total synthesis of fucosylated chondroitin sulfate (fCS) oligosaccharides and semisynthetic strategies to obtain fCS oligosaccharides and low molecular weight polysaccharides . The impMarine Drugs for their support and kind help.As guest editor, I am grateful to all the authors who contributed their excellent results to this Special Issue, all the reviewers who carefully evaluated the submitted manuscripts, and


Dolabella auricularia, contains three unique amino acid residues. Dol-10 can effectively induce apoptosis of lung cancer cells and other tumor cells at nanomolar concentration, and it has been developed into commercial drugs for treating some specific lymphomas, so it has received wide attention in recent years. In vitro experiments showed that Dol-10 and its derivatives were highly lethal to common tumor cells, such as L1210 leukemia cells (IC50 = 0.03 nM), small cell lung cancer NCI-H69 cells (IC50 = 0.059 nM), and human prostate cancer DU-145 cells (IC50 = 0.5 nM), etc. With the rise of antibody-drug conjugates (ADCs), milestone progress was made in clinical research based on Dol-10. A variety of ADCs constructed by combining MMAE or MMAF (Dol-10 derivatives) with a specific antibody not only ensured the antitumor activity of the drugs themself but also improved their tumor targeting and reduced the systemic toxicity. They are currently undergoing clinical trials or have been approved for marketing, such as Adcetris®, which had been approved for the treatment of anaplastic large T-cell systemic malignant lymphoma and Hodgkin lymphoma. Dol-10, as one of the most medically valuable natural compounds discovered up to now, has brought unprecedented hope for tumor treatment. It is particularly noteworthy that, by modifying the chemical structure of Dol-10 and combining with the application of ADCs technology, Dol-10 as a new drug candidate still has great potential for development. In this review, the biological activity and chemical work of Dol-10 in the advance of antitumor drugs in the last 35 years will be summarized, which will provide the support for pharmaceutical researchers interested in leading exploration of antitumor marine peptides.Dolastatin 10 (Dol-10), a leading marine pentapeptide isolated from the Indian Ocean mollusk  Some components possess ethylenic bonds and acetylenic bonds, which improve the stability and bioavailability of peptides . M. M1H NMRpartners . The modficantly .To sum up, this work was helpful to understand the structural characteristics of Dol-10 that played a role in microtubule polymerization and mitosis. The folding structure and high flexibility of Dol-10 together realized its excellent biological activity.®, as a typical representative of listed ADCs, has successfully overcome the obstacles in clinical application of Dol-10 and greatly promoted the research progress of ADCs. Moreover, in the process of systematical antitumor study of Dol-10, it is accidentally found that this antitumor peptide also has potent antifungal [Plasmodium falciparum [N- and C-terminal subunits, it can be predicted that more Dol-10 analogs based on modified central residues Dil, Dap, and Doe will appear soon. And there is no doubt that selecting appropriate monoclonal antibodies to couple with Dol-10 or its derivatives to prepare ADCs into the clinic is still one of the main research directions of Dol-10 in the future.Marine bioactive polypeptide Dol-10 has been investigated for more than 30 years, from its first discovery to the approval of the related drug for marketing, which brings a bright prospect for tumor treatment and is still one of the most active antitumor compounds found so far. Dol-10 and its derivatives can effectively inhibit the growth of tumor cells in vitro, but their clinical efficacy as a single drug in phase II clinical trials of solid tumors is frustrating due to adverse effects. For example, the most common peripheral neuropathy had brought a great blow to the clinical research of the marine peptide. Later, chemical researchers coupled monoclonal antibodies with Dol-10 derivatives by ADCs technology, and utilized the specificity of antibodies to transport drug molecules to target tissues for their functions. As a result, the systemic toxic and side effects of drugs were reduced, and the treatment window of drugs was improved. Adcetristifungal , antibactifungal , and antlciparum  biologic