PLoS ONE Research Articles

Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids.

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7–6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.

Development of an Electrochemical Biosensor of Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) for the Assessment of Traumatic Brain Injury (TBI)

INTRODUCTION Traumatic brain injury (TBI) is defined as a neurotrauma caused by a mechanical force applied to a head. An appropriate clinical therapy after TBI is crucial, or patients may suffer lasting effects in physical and mental abilities [1]. To find TBI, cranial computer tomography (CT) is commonly used, however, CT sometimes fails to find mild TBI (mTBI) even it accounts for majority of all cases of TBI. Recently, a blood-based biomarker, ubiquitin C-terminal hydrolase L1 (UCH-L1) has been regarded a promising marker for mTBI, as it becomes present in serum soon after TBI [2,3]. Actually, it was reported the elevated serum UCH-L1 level was observed with the soldiers exposed to low-level blast [4] and the athletes after sub-concussive dead hits [5]. Enzyme-linked immuno sorbent assay (ELISA) is currently used to detect the biomarker, however, since the ELISA is time-consuming procedure and requires specific laboratory equipment, development of a hand-held device to be used on-site in the field is expected. In this study, we propose and report a novel principle for a rapid, one-step electrochemical detection system for UCH-L1 to be dedicated for mTBI diagnosis. METHOD To detect UCH-L1, we chose an electrochemical method in terms of a rapid response time, miniaturization, and simple instrumentation. We employed a competitive assay combined with square wave voltammetry (SWV) on anti-UCH-L1 antibody immobilized electrode. In this system, a redox probe (phenazine ethosulfate: PES) modified UCH-L1 and the target biomarker is going to competitively bind to the antibody on the electrode, resulting electrochemical signal depend on the concentration of UCH-L1 in a sample. To construct the system, PES modified UCH-L1 was prepared using amine-reactive PES. This molecule has N-hydroxysuccinimide linked to PES, therefore, upon mixing amine-reactive PES with UCH-L1, it will react with amine group of lysine residue and make a covalent bond. After the modification, enzyme-linked immuno sorbent assay (ELISA) was performed to investigate the binding affinity of anti-UCH-L1 antibody against UCHH-L1 and PES modified UCH-L1. To detect UCH-L1, we added UCH-L1 together with PES-modified UCH-L1 on the antibody immobilized gold electrode, and performed SWV at a potential range of -0.5 to 0.1 V with different frequencies of square wave. RESUTLS The result of ELISA showed that the antibody had almost same affinity against non-labeled UCH-L1 and PES-modified UCH-L1. Therefore, the modification of PES did not affect the recognition of antibody. When we incubated the electrode with different concentration of PES-modified UCH-L1, a square wave voltammetry signal was increased in a concentration dependent manner, indicating the PES modified on the UCH-L1 was electrochemically active, and the amount of PES-modified UCH-L1 was detected by SWV when it was captured by anti-UCH-L1 antibody on the electrode. In the competitive assay, the incubation of intact UCH-L1 together with PES-modified UCH-L1 resulted in the SWV signal change depend on the UCH-L1 concentration. With this detection principle, we could detect UCH-L1 within 10 minutes without any washing procedure, which is ideal for the on-site detection of mTBI. CONCLUSION In this study, we developed a rapid electrochemical detection system of TBI biomarker, UCH-L1, based on the competitive of redox probe-modified UCH-L1 with the target. Using this detection system, we were able to observe SWV signal change within 10 minutes upon addition of UCH-L1. This study shows the proof-of-concept of this rapid competitive assay, and we hope it would help future development of TBI detection device. REFERENCES [1] S. W. Hoffman and C. Harrison, Clin. Neuropsychol., 23(8), 1400–1415 (2009).[2] R. Diaz-Arrastia, K. K. W. Wang, L. Papa, M.D. Sorani, J. K. Yue, A. M. Puccio, P. J. McMahon, T. Inoue, E. L. Yuh, H. F. Lingsma, A. I. R. Maas, A. B. Valadka, D. O. Okonkwo, G. T. Manley, TRACK-TBI Investigators, J. Neurotrauma, 31(1), 19–25 (2013).[3] L. Papa, G. M. Brophy, R. D. Welch, L. M. Lewis, C. F. Braga, C. N. Tan, N. J. Ameli, M. A. Lopez, C. A. Haeussler, D. I. M. Giordano, S. Silvestri, P. Giordano, K. D. Weber, C. Hill-Pryor, D. C. Hack, JAMA Neurol., 73(5), 551–560 (2016).[4] C. M. Tate, K. K. W. Wang, S. Eonta, Y. Zhang, W. Carr, F. C. Tortella, R. L. Hayes, G. H. Kamimori, J. Neurotrauma, 30(19), 1620–1630 (2013).[5] V. Puvenna, C. Brennan, G. Shaw, C. Yang, N. Marchi, J. J. Bazarian, K. Merchant-Borna, D. Janigro, PLoS ONE, 9(5), e96296–9 (2015).

Intranasal oxytocin decreases self-oriented learning.

Oxytocin has been found to play an important role in human social cognition and social interaction. Over the last two decades, surge studies have been conducted to investigate how oxytocin impacts other-oriented processes, such as trust and generosity (Zak et al. in PLoS ONE 2(11):e1128, 2007); however, the examination of the effect of oxytocin on self-related processes was relatively inadequate. Appropriate and efficient social interactions require both self- and other-related information processing. Recent studies have found that intranasal oxytocin (IN-OT) influences the self-related process, although the results have been mixed. The computational process underlying the effects of IN-OT on self-processing remains unknown. We aim to investigate the effect of IN-OT on self-oriented learning across different contexts (self-other independent vs. self-other dependent) and uncover the process by which IN-OT affects dynamic behavior changes. We performed two double-blind, placebo-controlled studies and used reinforcement learning theory to integrate action and related feedback for participants' behaviors. In study 1, IN-OT decreased self-oriented reward learning when self-oriented learning and prosocial (other-oriented) learning were assessed separately. These effects were partially due to the OT-related increase in choice variability during self-oriented learning. In study 2, IN-OT also decreased learning performance during self-oriented reward learning when self-related and other-related rewards were present together. These effects occurred at an early stage of the learning process and could be moderated by the participants' social value orientation. Our findings show that OT attenuates the process of self-oriented learning and provides an underlying computational process. Our findings shed new light on the dynamics of IN-OT's effects on human self-oriented learning processes. For future studies on OT effects on self-oriented learning, individual factors such as social value orientation should be taken into consideration in research development and analysis.

Gut Microbiota-Induced Regulatory T Cells in Patients with Hematological Malignancies Receiving Allogeneic Hematopoietic Stem Cell Transplantation: Towards Deciphering a Role for These Tregs in aGVHD

Prognosis for hematological malignancies treated with allogeneic stem cell transplantation (allo-HSCT) remains dismal and can often induce potentially deadly complications, such as graft versus host disease (GvHD). Steroids remain the first-line treatment for aGvHD. Nevertheless, new treatments are needed to improve allo-HSCT outcomes and reduce steroid complications. In this context, increasing evidences suggest that gut microbiota composition and the activity of regulatory T cells (Tregs) are involved in GvHD prevention (Elias and Rudensky, 2019, Br. J. Haematol. ; Shono and van den Brink, 2018, Nat. Rev. Cancer). We have identified a novel FoxP3-negative IL-10-secreting Treg subset, named DP8α, enriched in the colon mucosa and present in blood, characterized by its CD3+/CD4+/CD8αLOW/ CCR6+/CXCR6+ phenotype and its TCR-specificity for the gut ClostridiumIV bacterium Faecalibacterium prausnitzii (Sarrabayrouse et al., 2014, PLoS Biol ; Godefroy et al., 2018, Gastroenterology). Sizable fractions of these cells also expressed the membrane-bound ectonucleotidases CD39 and CD73 (gating strategy shown in Figure 1), which are directly involved in their suppressive activity in vitro (Godefroy et al., 2018, Gastroenterology). In vivo, these molecules play key regulatory roles both by degrading extracellular ATP and thus limiting its inflammasome-related proinflammatory role, and by inducing the production of immunosuppressive adenosine. Accordingly, an alteration in adenosine/purinergic signaling has been suggested in GvHD occurrence (Deaglio et al., 2007, J. Exp. Med. ; Wang et al., 2013, PLoS ONE). Thus, we postulated that F. prausnitzii-reactive DP8α Tregs, whose suppressive activity depends on purinergic signaling, could bridge microbiota dysbiosis and GvHD incidence in allo-HSCT patients. In support of this, decreased levels of Clostridium bacteria, especially Faecalibacteriumspp, in patient's stools, have been associated with aGvHD risk (Noor et al., 2019, J Innate Immun). Moreover, mice gavaged with butyrate-producing and Treg-inducing Clostridia displayed milder GvHD and improved survival (Mathewson et al., 2016, Nat. Immunol.). Therefore, we recently analyzed 63 patients with hematological malignancies, who received allo-HSCT, among which 21 developed an aGvHD (pathologies and treatments are listed Table I). We quantified circulating DP8α Tregs and analyzed their expression of CD39 and CD73 in donors, before and after mobilization, and in patients at one week before and 1, 2, and 3 months post allo-HSCT. Strikingly, results clearly showed that aGvHD development was strongly associated with a lack of expression of CD73 by DP8α Tregs (p<.0001) (Fig.2A), but not by any other T cell subsets analyzed. DP8α frequency and their CD39 expression were similar in GvHD+ versus GvHD- patients. Importantly, CD73 decrease did not result from their corticotherapy since it was equally observed before and after treatment (Fig.2B) and since in vitro treatment of PBMCs with methylprednisolone did not alter CD73 expression whatsoever. Therefore, the CD73 decrease observed in aGvHD patients, appears relevant and likely related to the aGvHD condition itself. It is noteworthy that this cohort was rather heterogeneous in terms of hematological diseases, conditioning regimens and prophylaxis treatments (Table I). Nevertheless, this drastic CD73 decrease in aGvHD patients was observed across all these conditions, strongly strengthening its relevancy. Altogether, these data strongly suggest that a CD73-dependent functional alteration of DP8α Tregs are, at least in part, involved in aGvHD occurrence. These results could therefore be used to predict GvHD risk and also give rise to the development of therapeutic strategies. Such innovative treatments could be based on the infusion of DP8α Tregs with appropriate features, since we have shown both the uniquely high proliferating potential and the stable immunosuppressive function of these cells in vitro. Moreover, administration of DP8α target antigens (F. prausnitzii-derived), in the form of peptides, proteins, or even bacterial lysates, such as probiotics, could also be foreseen to either directly in vivo expand these Tregs or indirectly through the induction of tolerogenic dendritic cells (Alameddine et al., 2019, Front Immunol) and subsequent Treg differentiation/priming to limit GvHD-related inflammation. Figure 1 Disclosures Chevallier: Incyte Corporation: Honoraria.

JAK2 V617F Mutation Negative Polycythemia Diagnosed By IGF-1R, EEC Formation , and IRS2 over Expressions

We previously reported that by flow cytometry study of mononuclear cells (peripheral blood) of IGF-1 R (insulin growth factor -1 receptor) can help in differentiating polycythemia vera (PV) from secondary erythrocytosis (PLoS ONE 11(11): e016529), in patients negative for JAK2V617F mutation who requires frequent phlebotomy. EEC (Endogenous erythroid colony formation) formation which was shown to be related to IGF-1 signaling was employed as a minor criteria of PV prior to 2016 WHO criteria for the diagnosis of PV. IRS-2, an IGF-1 activation downstream signaling protein was found to be a docking protein, specific for erythropoietin-induced erythropoiesis. Current studies were designed to correlation between IGF-1R over-expression to EEC (Endogenous erythroid colony formation) formation and IRS-2 over expression in those patients who required frequent phlebotomy with elevated IGF-1R and JAK2 mutation negative patients. Materials and Methods: Patients: 8 patients withsecondary polycythemia (JAK2V617F mutation negative), and 12 MPN patients (6 ET, 3 PV and 3 MF) were studied. Of the 12 MPN patients, 11 were JAK2 mutation positive and 1 was CALR mutation positive. All 20 patients had elevated IGF-1 R. Methods: 1) IGF-1R expression: 106 cells blood mononuclear cells were incubated with 10 μL of PE-conjugated control or anti-IGF1R IgG (R&D Systems) for 30 minutes. Fluorescent intensity was detected by a flow cytometry. 2) EEC formation: Erythroid colony cultures using Methyl cellulose based medium without (MethoCult H4230) or with pre-inclusion of cytokines (MethoCult H4230, including erythropoitin) from StemCell Technologies Inc. were used. On day 10-12, colonies were scored for Burst forming unit-erythroid (BFU-e), Colony forming unit-erythroid (CFU-e), and Colony forming unit cells (CFU-c) according to the manufacturer's graphic guide. 3) IRS-2 Expression: RT-PCR Predesigned primers for IRS-2 and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR blood mononuclear cells was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of IRS-2 was compared with each internal control and the final patient to normal control ratio was the average of the three. Results: 7 out of 8 patients with secondary PV, were found to have EEC formation. 12 MPN patients had increased expression of IRS-2 (Fig 1). 2 patients with secondary PV, so far studied also were found to have increased expression of IRS-2. Conclusion: 1) In patients with secondary PV (JAK2 mutation negative and increased IGF-1 and requiring frequent phlebotomy0, 7 out of 8 (87%) were found to have EEC formation. This suggests that IGF-1 by Flow cytometry can replace EEC as a supplement to the diagnosis of PV. 2) In JAK2 or CALR mutation positive patients , IGF-1R over-expression were found to have to have IRS-2 over-expression , 2 patients with secondary PV so far studied were also found to have elevated IRS-2 signaling. This suggests that the etiology of erythrocytosis in these secondary PV is likely through IRS-2 pathway. Further studies on this is still in progress. Disclosures No relevant conflicts of interest to declare.

Using data science for medical decision making case: role of gut microbiome in multiple sclerosis

BackgroundA decade ago, the advancements in the microbiome data sequencing techniques initiated the development of research of the microbiome and its relationship with the host organism. The development of sophisticated bioinformatics and data science tools for the analysis of large amounts of data followed. Since then, the analyzed gut microbiome data, where microbiome is defined as a network of microorganisms inhabiting the human intestinal system, has been associated with several conditions such as irritable bowel syndrome - IBS, colorectal cancer, diabetes, obesity, and metabolic syndrome, and lately in the study of Parkinson’s and Alzheimer’s diseases as well. This paper aims to provide an understanding of differences between microbial data of individuals who have been diagnosed with multiple sclerosis and those who were not by exploiting data science techniques on publicly available data.MethodsThis study examines the relationship between multiple sclerosis (MS), an autoimmune central nervous system disease, and gut microbial community composition, using the samples acquired by 16s rRNA sequencing technique. We have used three different sets of MS samples sequenced during three independent studies (Jangi et al, Nat Commun 7:1–11, 2016), (Miyake et al, PLoS ONE 10:0137429, 2015), (McDonald et al, Msystems 3:00031–18, 2018) and this approach strengthens our results. Analyzed sequences were from healthy control and MS groups of sequences. The extracted set of statistically significant bacteria from the (Jangi et al, Nat Commun 7:1–11, 2016) dataset samples and their statistically significant predictive functions were used to develop a Random Forest classifier. In total, 8 models based on two criteria: bacteria abundance (at six taxonomic levels) and predictive functions (at two levels), were constructed and evaluated. These include using taxa abundances at different taxonomy levels as well as predictive function analysis at different hierarchical levels of KEGG pathways.ResultsThe highest accuracy of the classification model was obtained at the genus level of taxonomy (76.82%) and the third hierarchical level of KEGG pathways (70.95%). The second dataset’s 18 MS samples (Miyake et al, PLoS ONE 10:0137429, 2015) and 18 self-reported healthy samples from the (McDonald et al, Msystems 3:00031–18, 2018) dataset were used to validate the developed classification model. The significance of this step is to show that the model is not overtrained for a specific dataset but can also be used on other independent datasets. Again, the highest classification model accuracy for both validating datasets combined was obtained at the genus level of taxonomy (70.98%) and third hierarchical level of KEGG pathways (67.24%). The accuracy of the independent set remained very relevant.ConclusionsOur results demonstrate that the developed classification model provides a good tool that can be used to suggest the presence or absence of MS condition by collecting and analyzing gut microbiome samples. The accuracy of the model can be further increased by using sequencing methods that allow higher taxa resolution (i.e. shotgun metagenomic sequencing).

Introducing the PLOS ONE Collection on the neuroscience of reward and decision making.

The survival of an organism depends on the ability to make adaptive decisions to achieve the needs of the organism: where to get food, who to mate with, and how to evade predators. Decision-making is a term used to describe a collection of behavioral and/or computational functions that guide the selection of an option amongst a set of alternatives. Some of these functions may include calculating the costs and benefits of a particular action, evaluating differences in value of each of the alternative outcomes and the likelihood of receiving a particular outcome, using past experiences to generate predictions or expectations about action-outcome associations, and/or integration of past experiences to make novel inferences that can be used in new environments. There is considerable interest in understanding the neurobiological mechanisms that mediate these decision-making functions and recent advances in behavioral approaches, neuroscience techniques, and neuroimaging measures have begun to develop mechanistic links between biology, reward, and decision making. This multidisciplinary work holds great promise for elucidating the biological mechanisms mediating decision-making deficits in normal and abnormal states. The multidisciplinary studies included in this Collection provide new insights into the neuroscience of decision making and reward.

Clinical Risk Factors for COVID-19 Among People With Substance Use Disorders.

Back to table of contents Previous article Next article DatapointsFull AccessClinical Risk Factors for COVID-19 Among People With Substance Use DisordersHefei Wen, Ph.D., Michael L. Barnett, M.D., M.S., Brendan Saloner, Ph.D.Hefei WenSearch for more papers by this author, Ph.D., Michael L. BarnettSearch for more papers by this author, M.D., M.S., Brendan SalonerSearch for more papers by this author, Ph.D.Published Online:6 Oct 2020https://doi.org/10.1176/appi.ps.202000215AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail As COVID-19 spreads across the United States, people with substance use disorder may be susceptible to contracting the disease and developing serious complications. COVID-19 epidemiology suggests that cardiovascular and chronic respiratory diseases and diabetes may increase the risk for severe illness (1). People with opioid use disorder and methamphetamine use disorder deserve particular attention, given the detrimental effects of long-term drug use on pulmonary function, other major organ systems, and the immune system (1).We used individual‐level data from the 2015–2018 National Survey on Drug Use and Health (NSDUH). Our sample consisted of three mutually exclusive groups of civilian, noninstitutionalized adults ages 18–64: those with past-year opioid or methamphetamine use disorder, those with other types of past-year substance use disorder (i.e., abuse of or dependence on alcohol, illicit drugs other than opioid and methamphetamine, or both), and those who did not have a past-year substance use disorder (2).We used logistic regression models to estimate differences among the groups in the overall and age-stratified rates of having an underlying condition that may heighten the risk for severe illness following COVID-19 infection. Models were adjusted for gender, race-ethnicity, and urban/rural residence. We used the Stata/SE “svy” procedure to account for NSDUH’s sampling weights and complex survey design (2).Nonelderly adults with opioid or methamphetamine use disorder were most likely to have an underlying condition (adjusted rate, 44.2%; p<0.001), compared with those with other types of substance use disorder (31.4%) and those without a past-year substance use disorder (33.5%). The higher rates of having an underlying condition among those with opioid or methamphetamine use disorder were observed consistently across all age categories (Figure 1).FIGURE 1. Adjusted rates of having clinical risk factors for COVID-19 severity and mortality among nonelderly U.S. adultsaaClinical risk factors recognized by the World Health Organization and the Centers for Disease Control and Prevention and included in the National Survey on Drug Use and Health questionnaire are cardiovascular disease, hypertension, chronic respiratory diseases, diabetes, cancer, chronic liver diseases, kidney disease, and HIV/AIDS. Rate estimates and 95% confidence intervals (whisker lines) were estimated with Stata/SE.Our findings underscore that opioid and methamphetamine use disorders are associated with underlying conditions identified as risk factors for COVID-19 severity and mortality. People with these conditions are an underserved group who may need additional support to ensure equal access to testing and treatment, especially as COVID-19 continues to impose unprecedented pressures on the nation’s health care system. Social distancing, self-quarantine, and other public health measures may disrupt their access to addiction treatment (2). Additional efforts are required to mitigate those unintended disruptions.Department of Population Medicine, Harvard Medical School, and Harvard Pilgrim Health Care Institute, Boston (Wen); Department of Health Policy and Management, Harvard T. H. Chan School of Public Health, Boston (Barnett); Department of Health Policy and Management, Johns Hopkins Bloomberg School of Public Health, Baltimore (Saloner). Tami L. Mark, Ph.D., and Alexander J. Cowell, Ph.D., are editors of this column.Send correspondence to Dr. Wen ([email protected]).The authors report no financial relationships with commercial interests.References1 Volkow ND. Collision of the COVID-19 and addiction epidemics. Ann Intern Med (Epub ahead of print, April 2, 2020).Crossref, Medline, Google Scholar2 2018 National Survey on Drug Use and Health: Methodological Summary and Definitions. Rockville, MD, Substance Abuse and Mental Health Services Administration, 2019. https://www.samhsa.gov/data/sites/default/files/cbhsq-reports/NSDUHMethodsSummDefs2018/NSDUHMethodsSummDefs2018.pdfGoogle Scholar FiguresReferencesCited byDetailsCited byBehavioral Health Workforce Development in the era of COVID-19: Examples From a State-Funded Intermediary Organization26 April 2022 | Community Mental Health Journal, Vol. 58, No. 8Experiences with substance use disorder treatment during the COVID-19 pandemic: Findings from a multistate surveyInternational Journal of Drug Policy, Vol. 101The Impact of the COVID-19 Pandemic on the Practice of Forensic Medicine: An Overview8 February 2022 | Healthcare, Vol. 10, No. 2Integrated vs nonintegrated treatment for perinatal opioid use disorder: retrospective cohort studyAmerican Journal of Obstetrics & Gynecology MFM, Vol. 4, No. 1Prevalence and correlates of SARS-CoV-2 seropositivity among people who inject drugs in the San Diego-Tijuana border region22 November 2021 | PLOS ONE, Vol. 16, No. 11 Volume 71Issue 12 December 01, 2020Pages 1308-1308 Metrics Keywordssubstance use disorderCOVID-19chronic diseaserisk factorPDF download History Received 2 April 2020 Revised 19 April 2020 Accepted 13 May 2020 Published online 6 October 2020 Published in print 1 December 2020

The influence of bioRχiv on PLOS ONE's peer‐review and acceptance time

AbstractThis study examines the relation between acceptance times in preprint publishing and journal publishing. Specifically, we investigated the association between a paper's posting time to bioRχiv, a preprints server, and journal articles' peer‐review and acceptance time for PLOS ONE. So far, of the total papers published in 1,626 academic journals, the average publication rate of those posted in bioRχiv is 40.67%. Meanwhile, PLOS ONE was the journal that published more papers. Analysis of peer‐review and acceptance time of papers published in journals via preprints showed the time these papers are posted in relation to these intervals. The median of the peer‐review and acceptance time of the journal submission date that was later than the date of first posting to bioRχiv was 110.00 days, and in the reverse case, it was 139.50 days. Posting to the preprint server before journal submission shows a better order than vice versa. This study provides us a good understanding of the peer‐review process. It also gives us good insights into optimizing this process, which would then facilitate paper publication and knowledge dissemination.

Handgrip strength cut-offs to define sarcopenia in Turkish population

Cruz-Jentoft AJ, Bahat G, Bauer J et al (2019) Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing 48:16–31. https://doi.org/10.1093/ageing/afy169 Article PubMed Google Scholar Bahat G, Tufan A, Tufan F et al (2016) Cut-off points to identify sarcopenia according to EuropeanWorking Group on Sarcopenia in older people (EWGSOP) definition. Clin Nutr 35:1557–1563. https://doi.org/10.1016/j.clnu.2016.02.002 Article PubMed Google Scholar Lauretani F, Russo CR, Bandinelli S et al (1985) Age-associated changes in skeletal muscles and their effect on mobility: an operational diagnosis of sarcopenia. J Appl Physiol 2003:1851–1860 Google Scholar Sallinen J, Stenholm S, Rantanen T et al (2010) Hand-grip strength cut points to screen older persons at risk for mobility limitation. J Am Geriatr Soc 58:1721–1726. https://doi.org/10.1111/j.1532-5415.2010.03035.x Article PubMed PubMed Central Google Scholar Ates Bulut E, Soysal P, Dokuzlar O et al (2020) Validation of population-based cutoffs for low muscle mass and strength in a population of Turkish elderly adults. Aging Clin Exp Res. https://doi.org/10.1007/s40520-019-01448-4 Article PubMed Google Scholar Dodds RM, Syddall HE, Cooper R et al (2014) Grip strength across the life course: normative data from twelve British studies. PLoS ONE 9:e113637. https://doi.org/10.1371/journal.pone.0113637 Article PubMed PubMed Central Google Scholar Download references As authors of this article: “Gulistan Bahat, Mehmet Akif Karan, Mustafa Altinkaynak” and the institution we work for, we received no grant, consuting fee or honorarium, support for any travel or other purposes, fees for participation in any review activities, payment for writing or reviewing, provision of writing assistance, medicines, equipment or administrative support, or any other. We have no relevant activities outside the submitted work as board membership, consultancy, employment, expert testimony, grants/grant pending, payment for any lectures, payment for manuscript preparation, patents, royalties, payment for educational activities, stock/stock options, travel/accommodation/meeting expenses unrelated to activities listed, or any other. There are no other relationships/conditions/circumstances that present a potential conflict of interest. Istanbul Medical School, Department of Internal Medicine, Division of Geriatrics, Istanbul University, Capa, 34390, Istanbul, Turkey Gulistan Bahat & Mehmet Akif Karan Istanbul Medical School, Department of Internal Medicine, Istanbul University, Capa, 34390, Istanbul, Turkey Mustafa Altinkaynak You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar Correspondence to Gulistan Bahat. On behalf of all the authors, the corresponding author states that there is no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors. For this type of study formal consent is not required. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions Bahat, G., Altinkaynak, M. & Karan, M.A. Handgrip strength cut-offs to define sarcopenia in Turkish population. Aging Clin Exp Res (2020). https://doi.org/10.1007/s40520-020-01704-y Download citation Received: 28 July 2020 Accepted: 31 August 2020 Published: 28 September 2020 DOI: https://doi.org/10.1007/s40520-020-01704-y

Other than detecting impact in advance, alternative metrics could act as early warning signs of retractions: tentative findings of a study into the papers retracted by PLoS ONE

The so-called altmetrics—short for alternative metrics—have gained their place in the scholarly publishing landscape, especially providing article-level complementary measures. But what do they point to? A hypothesis has found its way into the recent literature: they could be an early sign of impact, conveying in advance the information carried later by citations. Here the focus is on another possible relationship, namely, the one that may exist between alternative metrics and retractions. The research question is as follows: in comparison to non-retracted, contemporary publications, are the retracted articles likely to be more (or less) viewed and commented (but also «tweeted» and «blogged») before the retraction takes place? The above relationship is tested on a set of n = 209 papers retracted by PLoS ONE and a control group including 2n = 418 contemporary, non-retracted articles featured in the same journal. Significant and positive differences in means are found concerning the mentions in peer review sites and the number of views and downloads. On the whole, by considering five alternative metrics, about one-fourth of the retractions are predicted correctly.

Influence of Male Partners on HIV Disclosure Among South African Women in a Cluster Randomized PMTCT Intervention.

Disclosure of HIV serostatus is beneficial for women, their partners, and their infants as it enables women to actively participate in preventative care (Hodgson et al. in PLoS ONE 9(11):e111421, 2014; Odiachi et al. in Reprod Health 15(1):36, 2018). Therefore, it is important that interventions addressing HIV prevention include elements that foster disclosure of HIV to partners. This study conducted in South Africa utilizes the "Protect Your Family" (PYF) behavioral intervention and compares Prevention of Mother to Child Transmission (PMTCT) among women participating in the program versus those in a control program. Within both groups, male partners were either present or not present for the intervention. The purpose of this study was to examine differential disclosure over time for individuals in the different conditions and partner involvement. A firth logistic regression revealed an interaction in the experimental condition with male partners participating (b = - 2.84, SE = 1.56, p = .012), in which female participants were less likely to disclose their HIV status over time. Findings from this study illustrate that additional efforts are needed to empower women to disclose their HIV status.

Microvascular damage assessed by OCT Angiography for glaucoma diagnosis: a systematic review of the most discriminative regions.

Acta OphthalmologicaVolume 98, Issue 6 p. 572-572 ErratumFree Access Microvascular damage assessed by OCT Angiography for glaucoma diagnosis: a systematic review of the most discriminative regions This article corrects the following: Microvascular damage assessed by optical coherence tomography angiography for glaucoma diagnosis: a systematic review of the most discriminative regions Amerens Bekkers Noor Borren Vera Ederveen Ella Fokkinga Danilo Andrade De Jesus Luisa Sánchez Brea Stefan Klein Theo Walsum João Barbosa-Breda Ingeborg Stalmans Volume 98Issue 6Acta Ophthalmologica pages: 537-558 First Published online: March 16, 2020 First published: 20 August 2020 https://doi.org/10.1111/aos.14481AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Amerens Bekkers1,2#, Noor Borren1,2#, Vera Ederveen1,2#, Ella Fokkinga1,2#, Danilo Andrade De Jesus1,3, Luisa Sánchez Brea1, Stefan Klein1, Theo van Walsum1, João Barbosa-Breda3,4,5, Ingeborg Stalmans3,6 1Biomedical Imaging Group Rotterdam, Department of Radiology & Nuclear Medicine, Erasmus MC, Rotterdam, The Netherlands 2Clinical Technology, Delft University of Technology, Delft, The Netherlands 3Research Group Ophthalmology, Department of Neurosciences, KU Leuven, Leuven, Belgium 4Ophthalmology Department, Centro Hospitalar e Universitário São João, Porto, Portugal 5Faculty of Medicine, University of Porto, Porto, Portugal 6Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium Keywords: glaucoma, OCTA, review, erratum In the article, ‘Microvascular damage assessed by optical coherence tomography angiography for glaucoma diagnosis: a systematic review of the most discriminative regions’ by Bekkers et al. (2020), the results reported from the paper of Rabiolo et al (2018) were not extracted correctly. In their study, Rabiolo et al (2018) reported an analysis of patients with diabetic retinopathy and patients with glaucoma. The area under the receiver operating characteristic curves (AUROCs) reported for the patients with diabetic retinopathy were wrongly included in place of the AUROCs for discrimination of glaucoma from healthy eyes at the peripapillary region. While these changes affect some of the figures and tables displayed on the manuscript, they have a minor effect on the threshold used for quality assessment and, hence, they do not alter the subsequent analysis nor the conclusions presented in the review: the superficial layer at the peripapillary region is preferable for assessing glaucomatous vascular damage. References Bekkers A, Borren N, Ederveen V et al. (2020): Microvascular damage assessed by optical coherence tomography angiography for glaucoma diagnosis: a systematic review of the most discriminative regions. Acta Ophthalmol https://doi.org/10.1111/aos.14392Wiley Online LibraryPubMedWeb of Science®Google Scholar Rabiolo A, Gelormini F, Sacconi R et al. (2018): Comparison of methods to quantify macular and peripapillary vessel density in optical coherence tomography angiography. PLoS ONE 13: 1– 20. CrossrefWeb of Science®Google Scholar Volume98, Issue6September 2020Pages 572-572 ReferencesRelatedInformation

Driving eligibility: Implications of studies on seizure recurrence risk.

Driving is one of the most important issues for patients with seizures. The 2009 European directive provides a framework for evaluating standard situations in assessing the ability to drive. Such a framework may not be sufficient for individual scenarios. To analyse current data on seizure recurrence risks (RcRs) focusing on their potential implications for car driving issues (group 1). We evaluated current studies and meta-analyses on RcR. A meta-analysis of seizure-free patients who withdrew their medication (Lamberink et al Lancet Neurology 2017;16:523) created a nomogram and a web-based tool that allow estimating RcR in individual patients and thus to identify those in whom medication withdrawal is possible without the common driving ban during withdrawal. The 2-year prediction model of that meta-analysis has been recently externally tested and confirmed. A meta-analysis of patients with a first unprovoked seizure (Bonnett et al PloS ONE 2014;9:e99063) determined to which extent RcRs depend on established risk factors. The seizure-free period required to restart driving could be tailored according to the individual RcR. These current studies allow estimating individual RcR more precisely and thus modifying periods of driving bans beyond the existing guidelines.

Corrigendum

People and NatureVolume 2, Issue 4 p. 1251-1251 CORRIGENDUMOpen Access Corrigendum This article corrects the following: Wild assumptions? Questioning simplistic narratives about consumer preferences for wildlife products Amy Hinsley, Michael 't Sas-Rolfes, Jana McPherson, Volume 2Issue 4People and Nature pages: 972-979 First Published online: June 30, 2020 First published: 14 August 2020 https://doi.org/10.1002/pan3.10144AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The authors would like to correct an error in the third paragraph of the first section of this article (Hinsley & 't Sas-Rolfes, 2020), which references Crudge, Nguyen, and Cao (2018) as stating that wildlife farming should be banned. Crudge et al. (2018) in fact do not state that wildlife farming should be banned. To correct this, the sentence that reads: For example, findings of expressed preferences for ‘wild’ have been used to strongly recommend that farming has not been a success and should be banned (Crudge et al., 2018), or that existing bans should be maintained (Gratwicke et al., 2008). should be replaced with the following: For example, findings of expressed preferences for ‘wild’ have been used to conclude that farming of certain species has no conservation benefit (Crudge et al., 2018) and that existing bans on farming should be maintained (Gratwicke et al., 2008). REFERENCES Crudge, B., Nguyen, T., & Cao, T. T. (2018). The challenges and conservation implications of bear bile farming in Viet Nam. Oryx, 54(2), 252– 259. https://doi.org/10.1017/S0030605317001752 Gratwicke, B., Mills, J., Dutton, A., Gabriel, G., Long, B., Seidensticker, J., … Zhang, L. I. (2008). Attitudes toward consumption and conservation of tigers in China. PLoS ONE, 3, e2544. https://doi.org/10.1371/journal.pone.0002544 Hinsley, A., & 't Sas-Rolfes, M. (2020). Wild assumptions? Questioning simplistic narratives about consumer preferences for wildlife products. People and Nature. https://doi.org/10.1002/pan3.10099 Volume2, Issue4Special Features: Consuming wildlife – managing demand for products in the wildlife trade & Managing forest regeneration and expansion at a time of unprecedented global changeDecember 2020Pages 1251-1251 ReferencesRelatedInformation

Abstract 4222: Tucatinib, a selective small molecule HER2 inibitor, is active in HER2 mutant driven tumors

Abstract Tucatinib is an investigational, orally dosed, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2 without significant inhibition of EGFR. In this report, we describe the activity of tucatinib alone and in combination with trastuzumab in multiple HER2 mutant driven PDX models. Tucatinib was tested in 6 HER2 mutant PDX tumor models derived from colorectal, non-small cell lung cancer, gall bladder and gastric cancers with L755S, V77L or S310Y mutations. MCF-10A cells were engineered to express the L755S and V777L HER2 activating point mutations and the potency of tucatinib was evaluated in each of these cell lines using HER2 autophosphorylation as an endpoint. The activity of tucatinib was also evaluated in G776V_G/C and G776_YVMA in frame exon 20 insertion mutations, including the G776V_G/C mutant non-small cell lung cancer line NCI-H1781 using both cellular signaling and cell proliferation as endpoints. Tucatinib significantly inhibited tumor growth in 5 of the 6 HER2 point mutant PDX models, whereas trastuzumab alone significantly inhibited tumor growth in 1 of 6 models and the combination of both drugs was significantly active in all HER2 mutant models. Tucatinib inhibited the phosphorylation of HER2 in a L755S mutant MCF-10A cell line and inhibited the growth of 3 L755S mutant tumor models derived from colorectal, gastric and non-small cell lung cancer. Tucatinib also potently inhibited the phosphorylation of HER2 in a V777L mutant MCF-10A cell line, and in a colorectal PDX model containing a V777L mutation, tucatinib alone or in combination with trastuzumab induced tumor regressions. In two PDX models containing the S310Y extracellular domain mutation of HER2, tucatinib alone, or in combination with trastuzumab, resulted in tumor growth delays. In exon 20 insertion mutant derived cell lines, tucatinib inhibited HER2 phosphorylation, but not EGFR phosphorylation, and inhibited the phosphorylation of HER2, HER3, Erk1/2, MEK1 and AKT in the G776V_G/C HER2 mutant NCI-H1781 cell line. These data demonstrate that tucatinib, a highly potent and selective inhibitor of HER2 signaling without significant inhibition of EGFR, is active in multiple HER2 mutant driven tumor models. Importantly, tucatinib is active in models containing the L755S HER2 mutation which has been associated with lapatinib resistance preclinically (1), and trastuzumab resistance clinically (2). Taken together, these results support the idea that HER2 selective inhibition by tucatinib can block oncogenic signaling elicited by mutant HER2, and that it could potentially provide clinical benefit in patients with cancers with activating HER2 mutations.

Abstract 1543: Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery

Abstract World-wide, metastasis continues to be the leading cause of death in cancer patients1. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the progression of cancer metastasis and drug resistance, their mechanistic pathway and specific role in disease progression is not fully understood2. As such, the development of a reporter line that enables the real-time monitoring of the changing status of cells will not only aid in dissecting the EMT/MET pathway in the research field, but could also become a robust platform for new cancer drug development. E-cadherin, an adhesion protein expressed in epithelial cells, is upregulated in pancreatic cancer cells during MET and is associated with an increase in tight junctions and apico-basal polarity, as well as a change in morphology3,4. Here, we developed a novel PANC-1 ECAD-EmGFP reporter line using CRISPR/Cas9 genome-editing technology. In this cell line, the emerald green fluorescent protein (EmGFP) reporter was incorporated into the last exon of the endogenous E-cadherin gene, enabling real-time monitoring of MET status in live cells. The ECAD-EmGFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Functional data revealed that miRNA-200 treatment induced the increased expression of ECAD-EmGFP, and decreased expression of Snail, a transcription factor associated with mesenchymal traits. These changes in marker gene expression, as well as the decrease in invasive capacity upon induction, suggest that cells have undergone MET. This cell line is a valuable tool for dissecting the molecular mechanisms underlying EMT and MET and for evaluating or screening compounds targeting EMT and MET in pancreatic cancer. 1. Dizon DS, et al. Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011, 2016. 2. Nieto MA, et al. EMT: 2016. Cell 166: 21-45, 2016. 3. Polireddy K, et al, Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLOS ONE 11(10): e0164811, 2016 4. Wong S, et al. E-cadherin: Its dysregulation in carcinogenesis and clinical implications. Critical Reviews in Oncology / Hematology 121: 11- 22, 2018 Citation Format: Diana Douglas, Sangeeta Kumari, Jarkko Huuskonen, Weiguo Shu. Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1543.

Abstract LB-314: Development of reference material guaranteed copy number of DNA for liquid biopsy

Abstract cfDNA (cell free DNA) analysis using liquid biopsy plays an important role in cancer research. In particular NGS (next-generation sequencer)-based assay has attracted wide interests, because it can not only screen many cancer genes but also can detect ultra-low-concentration mutations at the same time. However, there is no reference material to evaluate correctly its performance. Currently, most researchers use diluted human genomic DNA that is commercially available as reference, but adjusting its concentration is difficult because of the Poisson distribution. Herein, we have developed a method to dispense cells with counting a cell one by one using inkjet technology (Manabu Seo, et. Al., 2019, Anal. Chem.). By lysing cells after dispensing, the defined copy number of DNA is prepared with a single molecule level. In this study, we assessed the feasibility of our reference material prepared by inkjet as a control for rare mutation detection (e.g. minimal sampling bias and high reproducibility). Spike-in control samples with exact allele frequencies with 0.07%, 0.53%, 0.64%, and 1.0%, were prepared by accurately dispensing with inkjet the cells that contain a sequence of exons 18, 19, 20, and 21 of EGFR. The EGFR sequence has G719A, E746_A750del, T790M, C797S, L858R, and L861Q mutations. For comparison, a few samples were also prepared by manual dilution process. The EGFR mutations were analyzed by the method of high-sensitivity procedure using NGS (Yoji Kukita, et. Al., 2013, PLoS ONE.). Result of the inkjet method had less variation than the manual dilution method, and high repeatability was obtained. Especially in E746_A750del (0.53%) the inkjet samples were tested three times (n = 24 each), and their CVs (coefficient of variation) were 17%, 17%, and 12%. In contrast, the manual sample's one was 33%. In addition, linearity of T790M and L858R were R2 = 0.98 and R2 = 0.96, respectively. The reference material produced by inkjet method can be provided under the same conditions at each facility and make it possible to improve the reliability of liquid biopsy. Therefore, the reference material could be appropriate standard for external quality control for EGFR mutant detection of liquid biopsy. Citation Format: Yusuke Osaki, Yoshiharu Sato, Miho Ishii, Yuki Yonekawa, Ryo Matoba. Development of reference material guaranteed copy number of DNA for liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-314.

Abstract 1476: Vessel normalization following LDH-A knockdown in murine breast tumors

Abstract Abnormal vessel formation plays a major role in tumor progression. Previously, we showed that lactate dehydrogenase A (LDH-A) knockdown (KD) reduces orthotopic 4T1 breast tumor lactate, and has a robust anti-tumor effect through changes in the tumor microenvironment (TME) [1, 2]. Tumors with LDH-A downregulation have lower VEGF-A secretion and reduced formation of new blood vessels [2]. In addition, LDH-A KD cells/tumors displayed reduced HIF-1 signaling, as assessed by BLI using HRE-exGLuc reporter. Changes in the bioluminescence signal corresponded with changes in HIF-1α staining. LDH-A KD tumors also showed less HIF-1α staining compared to control tumors, consistent with less pimonidazole (hypoxia) staining [2]. Murine 4T1 breast cancer cells and tumors with LDH-A knockdown were developed and described [2]. To evaluate the degree of tumor neovascularization and its normalization, we stained tumors for CD31 (endothelial marker) and NG2 (pericytes marker). For pericyte coverage quantification, vessels were counted, and percentage of vessels covered by pericytes were calculated. Blood vessel permeability was assessed by intravenous injection of Evans Blue dye and tumor dye extravasation was quantified. To assess in vivo vascular network over time, a raster-scan optoacoustic mesoscopy (RSOM) was performed. Vessel structure in 4T1 murine tumors changes significantly following LDH-A KD. Normalized LDH-A KD tumor neovascularization was shown using the double immunofluorescence staining for CD31 and NG2, including a more normal vascular morphology, with shorter, more linear (less branched) vessel structure, with a higher percentage of vessels covered by pericytes compared to control tumors. Evans Blue dye extravasation was significantly less (lower vascular permeability) in LDH-A KD tumors compared to control tumors. Applying RSOM to follow the development of the vascular network during tumor growth, we showed better vascularization of LDH-A KD tumors. Furthermore, the number of circulating tumor cells (CTCs) was also significantly lower in blood collected from mice bearing LDH-A KD tumors. We show that LDH-depletion leads to tumor vessel normalization that includes several unique features: structural changes, greater coverage of blood vessel by pericytes and reduced vessel leakiness, leading to changes in the tumor microenvironment, inhibition of metastases formation and prolongation of survival. 1. Rizwan, A., et al., Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors. Clin Cancer Res, 2013. 19(18): p. 5158-69. 2. Serganova, I., et al., LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. PLOS ONE, 2018. 13(9): p. e0203965. Citation Format: Inna Serganova, Kiranmayi Vemuri, Ivan Cohen, Matthew Lubin, Sheryl Roberts, Masatomo Maeda, Mayuresh Mane, Henry Mann, Thomas Reiner, Eric Chan, Dmitry Yarilin, Katia Manova-Todorova, Roberta Zappasodi, Taha Merghoub, Jason Koutcher, Ronald Blasberg. Vessel normalization following LDH-A knockdown in murine breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1476.

Abstract 3687: Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells

Abstract ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic surface antigen that is expressed on breast cancer, but not on normal postpartum tissues. We found ROR1 was a receptor for Wnt5a, and high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). More recently, ROR1 could activate RhoGTPases and enhance tumor growth in breast cancer (Zhang S., et al., PNAS, 116(4), 2019). Such effects of Wnt5a on breast cancer patient-derived-xenograft (PDX) tumors could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. However, the molecular mechanism(s) for how ROR1 contributes such functions remained elusive. We performed mass spectrometry-based proteomic analysis on anti-ROR1 immune precipitates from breast cancer PDX tumors and detected DOCK1 in addition to ROR1, implying that ROR1 associates with DOCK1. DOCK1 (Dedicator of cytokinesis 1, also known as DOCK180) that expressed in breast cancer PDXs and cell lines, is a cytoplasmic protein, and a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1. Moreover, DOCK1 consists SH3 domain, which allows it to bind characteristic motifs (-P-X-X-P-), which often are found in the proline rich domains (PRD) of other proteins. Immunoblot analysis confirmed that ROR1 associated with DOCK1 in breast cancer PDX tumors or MCF7 cells transfected to express ROR1 (MCF7-ROR1) in response to Wnt5a, leading to enhanced activation of Rac1. To explore functional role, we performed a 2-week colony-formation assay using MCF7-ROR1 cells, and found that treatment of Wnt5a could enhance number of colonies of MCF7-ROR1 cells and that such colony formation capability could be inhibited by reducing expression of DOCK1 with specific siRNA, but not by control siRNA, suggesting a dependency on DOCK1 for Wnt5a-enhanced MCF7-ROR1 cells colony formation capability. Moreover, such effects of Wnt5a to enhance colony formation ability of MCF7-ROR1 cells could be inhibit by cirmtuzumab. We also introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1 proline-rich-domain (PRD) at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P⇒A mutants, ROR1P(808)A was unable to recruit DOCK1 to ROR1, or activate Rac1 in response to Wnt5a. Next we examined the tumor-growth potential of MCF7-Ctrl, MCF7-ROR1, or MCF7-P808A cells. The cells (5 × 104 cells/inoculate in RPMI 1640 together with an equal volume of Matrigel) were injected subcutaneously into the second right mammary pad of 4-to 6-week-old Rag2−/−γc−/- mice pretreated with estrogen pellets (17β-estradiol), which were placed subcutaneously in the intrascapular region one week before the cells inoculation. We found that MCF7-ROR1 cells (n=6) had significantly higher tumor growth relative to MCF7 cells (n=8), which had the same tumor growth as that of MCF7-P808A cells (n=6). Moreover, MCF7-ROR1P808A tumors had less activation of Rac1 compared to MCF7-ROR1 cells expressing wild-type ROR1. This study reveals that the recruitment of DOCK1 to ROR1 may be critical for the capacity of Wnt5a to enhance breast cancer tumor growth. Citation Format: Md Kamrul Hasan, George F. Widhopf II, Suping Zhang, Sharon M. Lam, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3687.