Evolving in sync with the computation revolution over the past 30 years, computational biology has emerged as a mature scientific field. While the field has made major contributions toward improving scientific knowledge and human health, individual computational biology practitioners at various institutions often languish in career development. As optimistic biologists passionate about the future of our field, we propose solutions for both eager and reluctant individual scientists, institutions, publishers, funding agencies, and educators to fully embrace computational biology. We believe that in order to pave the way for the next generation of discoveries, we need to improve recognition for computational biologists and better align pathways of career success with pathways of scientific progress. With 10 outlined steps, we call on all adjacent fields to move away from the traditional individual, single-discipline investigator research model and embrace multidisciplinary, data-driven, team science. Do you want to attract computational biologists to your project or to your department? Despite the major contributions of computational biology, those attempting to bridge the interdisciplinary gap often languish in career advancement, publication, and grant review. Here, sixteen computational biologists around the globe present "A field guide to cultivating computational biology," focusing on solutions. Fig 1).Biology in the digital era requires computation and collaboration. A modern research project may include multiple model systems, use multiple assay technologies, collect varying data types, and require complex computational strategies, which together make effective design and execution difficult or impossible for any individual scientist. While some labs, institutions, funding bodies, publishers, and other educators have already embraced a team science model in computational biology and thrived –7, otherBox 1).In this article, we define computational science extremely broadly to include all quantitative approaches such as computer science, statistics, machine learning, and mathematics. We also define biology broadly, including any scientific inquiry pertaining to life and its many complications. A harmonious deep integration between biology and computer science requires action—we outline 10 immediate calls to action in this article and aim our speech directly at individual scientists, institutions, funding agencies, and publishers in an attempt to shift perspectives and enable action toward accepting and embracing computational biology as a mature, necessary, and inevitable discipline .Many high-impact papers have computational biologists in key authorship positions. In biology journals, these are customarily the first and last positions. Middle-author placements are very common for computational biologists, reflecting a methodological contribution on a paper to address a particular biological question. Co-first or co-senior authorships provide a means to provide credit when computational contributions are equally important to a paper, but such designations are often dramatically discounted in grant, hiring, promotion, and tenure assessments. For example, we have seen recent comments such as: “No recent first or senior author papers were listed ” . Other author ordering alternatives are possible, and scientific publishing has experienced dramatic change in recent years . One optFig 2). Institutions can address this by altering promotion structures to depend upon an author’s contributions, regardless of order in the publication. One innovative solution was undertaken in 2021 by the Australian Research Council (ARC): A 500-word section was added to proposals for applicants to describe “research context” and “explain the relative importance of different research outputs and the expectations” in the specific discipline/s of the applican10 ten most relevant scientific results  and verifiable description of the impact of these items . The Royal Society Resume for Researchers was also designed to support and highlight a variety of research contributions [Fundamentally, these issues will only be solved through educating institutional leaders and grant/paper reviewers on how to best understand and appreciate different kinds of scientists’ contributions. From calling computational biologists “research parasites”  to “mathibutions . This prAnother complication to evaluating computational biologists is that their primary research output may not be papers but instead valuable software or data . The US Schemes for tracking the usage of software and data are a work in progress . CitatioQuality software development and maintenance is crucial for efficient and reproducible data analysis and is a key ingredient for successful computational biology projects . CommuniWet lab biologists trained in traditional evaluation schemes can be quick to dismiss a researcher with a lack of a single driving biological question for the laboratory, many middle-authored papers, publication in computational conferences rather than journals, a low citation count or h-index (due to field-specific differences), or funding through grants led by others, leading to comments such as “How do people like you ever get last-author papers?” and “an overly strong reliance on collaborators” . ComputaWe hope to reach a day where quantitative skills are so pervasive  that calling someone a “computational biologist” sounds just as odd as a “pipette biologist” . In the Likewise, institutions should take care to ensure that interdisciplinary researchers are recognized and rewarded for contributions across disciplines and departments, for example, through the evaluation system, additional compensation, and supplemental administrative staff. These researchers face more than their fair share of demands from collaborative roles on grants, administrative leadership, educational initiatives, thesis committees, and consultations. Many are jointly appointed to 2 or more departments, introducing additional service requirements that are invisible to each individual department . ComputaAs large datasets become increasingly common, computational expertise is a necessary asset for any biologist. The deepest insights often result from data analyzed by the biologists who designed and conducted the experiment. Likewise, an understanding of biological data is a necessary asset for any computer scientist working on biological data. The most impactful methodological leaps often result from a computer scientist with a deep understanding of the nuances and limitations of particular data. Institutions can help hybrid trainees bridge gaps through computational biology training, strategic organization of physical space, and team science–oriented evaluation metrics for mentors.The first step in acquiring practical computational biology skills is to become comfortable with the basics in the unfamiliar domain: either programming or biology. Societies and other nonprofits can play a major role here; examples include the iBiology, Software Carpentries, CABANA in Latin America, and NEUBIAS in Europe –25. StilFig 3).Institutions can also organize spaces to facilitate deep integration between biology and computer science. Combining wet and dry lab spaces encourages interactions among researchers with diverse expertise. For computational biology trainees who are embedded in laboratories with a focused and single-discipline research agenda, institutions and lab heads can seek mentors from complementary domains, and these external mentors should be rewarded institutionally. We urge institutional oversight for trainees hired by individual out-of-discipline PIs to be sure that they are learning in their chosen field and not treated as inexpensive hired hands. We also emphasize that not all labs require supplemental trainee supervision and mentorship; many lead investigators in computational biology are quite experienced enough to cover both sides  is essential to modern biomedical data science and requires compute power, data storage, networking, and system administration—all of which can introduce significant costs to research projects. For most institutions, this computing infrastructure is often not centrally provided nor well supported; it often falls through the cracks between institutional information technology (IT) and research offices. As a result, individual laboratories often independently (and inefficiently) fund their own infrastructure and perform systems administration for clusters or the cloud that are far outside their actual expertise. Institutions that wish to foster computational research should subsidize infrastructure costs and provide support staff to assist in its use. Some funding agencies already offer grant mechanisms that support computing hardware or cloud-based computing, such as the US STRIDES initiative, but others should also recognize these necessary costs and increase budgets to better support computational biology.Historically, computational biologists relied on collaborators or public resources for datasets. It is now becoming more common for researchers with an informatics background to be running experiments, whether as a primary data source or to benchmark new technologies, validate algorithms, and test predictions and theories. Being able to bridge the dry lab–wet lab gap can have a major impact on a computational biologist’s success. Institutions and laboratory neighbors can flexibly offer laboratory space for computational biologists when needed, and they can offer appropriate biology mentors for computational biologists joining computational groups. Core facilities can extend support: for example, rather than only ingesting fully prepped samples or only providing instrument time, facilities can offer full service (from sample prep to data acquisition) to computational laboratories for an additional fee.“Data available upon request” is outdated, ineffective , and shoExplicit funding mechanisms aimed at data reuse can also facilitate algorithm development. These mechanisms can range from grant applications specifically targeting dataset reuse, a hackathon hosted by a disease foundation, or even a DREAM challenge in which the organizers curate a dataset specifically for algorithm development. These exercises also encourage the next generation of researchers to challenge, or augment, conclusions reported in original marker papers. Institutions can also fund internal algorithm development efforts to improve analyses of commonly generated data.Data-generating biologists can share data types through a growing ecosystem of repositories . LogistiInstitutions with large medical centers are realizing the promise of discovery from multimodal datasets of their unique patient cohorts, including primary clinical data as well as data from corresponding biospecimens using emerging molecular and imaging technologies. For example, deep learning has the potential to revolutionize pathology, but sufficient data are needed, often thousands of annotated images spanning patient groups. Infrastructural, ethical, and cultural considerations all place barriers to large-scale data access for computational research.Institutions can play a role in supporting data access by providing centralized database structures that automatically ingest patient electronic health records and research-level data collected on these patients . This reThe current system rewards scientists and institutions that closely guard clinical datasets, because exclusive analysis can benefit careers and the data can be monetized due to commercial demand for novel biomarkers and therapeutic targets. Strong leadership is essential to incentivize academic investigators to collect datasets in a coordinated way across disease groups to enable research for public benefit. Federated learning, where machine learning models can be trained on multiple datasets without actually sharing the raw data, may work around some barriers.Visionaries a decade ago aspired to bridge the domains of computational sciences and biology –7. SinceAs computational biology has grown rapidly over the last 30 years, we may ask what computational biology will look like 30 years from now, if cultures shift in the ways we propose.We foresee the ever-growing amount of data and associated analytical questions outstripping the supply of researchers with computational skills. This unmet demand will drive wet lab biologists to use software, which funding bodies and publishers should support to become more optimized, reproducible, maintainable, and easy to use without relying on a dedicated computationalist. Likewise, as more data become open and interoperable and as contracted wet lab facilities grow, computationalists may not need to rely on dedicated wet lab scientists.In the interim, as the field continues to develop new methods and biological data types, we foresee research parasitism and team science collaborations flourishing, and the scientists and institutions who focus most on cultivating computational biology will be rewarded.Over time, the distinction between wet and dry biologists may fade, as both are working toward a common goal of understanding biology, and hybrid biologists ,33 will


Recent studies demonstrated neural systems in bilateral fronto-temporal brain areas in newborns specialized to extract linguistic structure from speech. We hypothesized that these mechanisms show additional sensitivity when identically structured different pseudowords are used communicatively in a turn-taking exchange by two speakers. In an fNIRS experiment newborns heard pseudowords sharing ABB repetition structure in three conditions: two voices turn-takingly exchanged different pseudowords (Communicative); the different pseudowords were produced by a (Single Speaker); two voices turn-takingly repeated identical pseudowords (Echoing). Here we show that left fronto-temporal regions (including Broca’s area) responded more to the Communicative than the other conditions. The results demonstrate that newborns’ left hemisphere brain areas show additional activation when various pseudowords sharing identical structure are exchanged in turn-taking alternation by two speakers. This indicates that language processing brain areas at birth are not only sensitive to the structure but to the functional use of language: communicative information transmission. Newborns appear to be equipped not only with innate systems to identify the structural properties of language but to identify its use, communication itself, that is, information exchange between third party social agents—even outside of the mother–infant dyad. These regions show higher activation for ABB structured pseudowords (“mu-be-be”) than to random ABC controls (“mu-be-ga”), since such repetitions could hint at abstract rule-like structures, indicative of human language4. Such activations may reflect early precursors of syntactic abilities emerging already in infancy7.Humans are a highly social species, adapted to acquire language, our species-unique capacity enabling communicative information transmission between cooperative partners. Recent research uncovered neural systems in the newborn brain, mainly in left inferior frontal areas, involving Broca’s area, dedicated to language processing in adults8 and preparedness for ostensive-inferential communication9. 10.5-month-old infants attribute agency to unfamiliar entities who produce turn-taking exchanges of variable signal sequences10 and 13-month-olds assume that exchanging variable signal sequences—as opposed to repetition of identical signals—transmit information11. Importantly, these studies identified two necessary criteria for infants to recognize communicative information transfer. First, identifying two agents, who are taking interactional turns in producing signal sequences. Second, recognizing that the signal sequences exchanged vary sufficiently to potentially transfer novel information between the agents. The latter reflects the information theoretical constraint that by exchanging identical signals it is not possible to transmit information12. These two lines of research may demonstrate separate cognitive adaptations, however, recognizing communication and information exchange may provide a key context for acquiring the structural properties of language already from birth.A separate line of research demonstrated preverbal infants’ sensitivity to specialized ostensive signals that highlight communicative intentions4 and used sequences of pseudowords that shared an ABB structure to create three conditions. In our main, Communicative condition, a female and a male speaker took turns in uttering different ABB tokens . In the Single Speaker control condition, in each block either a male or a female voice uttered various ABB sequences , providing the same amount of syllabic variability as the Communicative condition. In the Echoing condition, a male and a female speaker took turns in repeating identical pseudowords continuously . We predicted that if newborns are sensitive to both criteria of communicative information exchange, that is, the presence of two speakers and the variability of exchanged tokens, they should respond more strongly to the Communicative condition than to the two control conditions.Here we use functional near-infrared spectroscopy (fNIRS) to test the hypothesis that newborns’ neural systems dedicated to extracting linguistic structure are integrated with their preparedness to recognize the functional use of language for communicative information transmission between agents. We built on a previous fNIRS study13 show that the newborn brain responded to all three conditions significantly more than to baseline  as a within-subject factor over oxyHb concentration changes revealed a significant main effect of Condition over the bilateral fronto-temporal areas, including channels 2 and 4 in the LH (p < .001) and channels 19 and 22 in the RH (p < .001) . The Communicative condition also evoked stronger responses than the Single Speaker condition in the LH over channel cluster 1, 4, 6 (p < .001). Additionally, stronger activity was found for the Single Speaker than for the Echoing condition over the RH channel cluster 14 and 17 (p < .001), whereas the opposite pattern was observed in RH channel 19 (p < .001), although neither differed from the Communicative condition. A similar analysis over deoxyHb did not show significant results, as is usual with infant fNIRS data.The hemodynamic responses are shown in Fig. 14: (A) two agents taking interactive turns, where (B) the exchanged signals contain variability. The Single Speaker condition presented the same sequence of different pseudowords, but lacked turn-taking between two agents, while the Echoing condition involved two speakers, who, however, repeated identical pseudowords with no variation. We found that relative to the other two conditions, the Communicative condition evoked increased activation in the left fronto-temporal areas of newborns' brain, which have been shown to subserve language processing in humans form birth15.In an fNIRS experiment we demonstrated that left fronto-temporal areas of the newborn brain show increased activation to structured speech when it is communicatively exchanged by two voices in turn-taking alternation, with the potential for information transmission. Only our Communicative condition met both criteria for communicative information exchange16. The very brain region that is activated by sequences with repetitive (ABB) as opposed to random structured pseudowords (ABC)18 showed additional activation to the ABB baseline of the Single Speaker condition, when varying ABB structured pseudowords were exchanged by two voices in the Communicative condition. This outcome suggests that neural systems dedicated to language acquisition serve not only the extraction of linguistic structures, but recognize the communicative, information transmitting use of language. Importantly, this effect is not simply social: if two agents take turns using linguistic structures interactively but merely repeat each other (Echoing condition), these systems are not activated. Newborns could have shown lower neural activity to turn-taking repetitions of identical linguistic stimuli because perfect predictability and lack of signal variability were not compatible with transmission of novel information.The significance of the present finding is that increased neural activation to communication was observed in left fronto-temporal regions, including Broca’s area, which are specialized to respond to linguistic structures, not to processing acoustic features20, we did not intend to investigate the origins of language, and the potentially innate mechanisms we identified are not derived from any particular theory of how language evolved. In fact, our results are just as well compatible with language models that are not motivated by its communicative use but by the productivity of thought21; such an account is not precluded by models of ostensive inferential communication22. Inferential models of communication23 suggest that the kind of communication humans engage in is specific to our species24, and human infants are indeed sensitive to communicative ostensive-referential cues25 including contingent reactivity26. In our study, even though all stimuli were perfectly contingent temporally, for which newborns may have a preference27, their brain activated less to a perfectly contingent exchange of contents (Echoing) and more to a high-but-imperfect level of contingency, structurally identical yet variable tokens (Communication), as if they sought to extract more information from such imperfectly contingent signal sequences. In a broader sense, Echoing may be taken to be a special kind of communicative exchange , which, in non-continuous form, typically addresses the recipient by expressing communicative intent that the recipient accepts. Yet our results show that newborns have a preference for higher communicative complexity, which enables them to recognize potential information transfer. In sum, no current nativist model appears to predict that any kind of sensitivity to communicational pragmatics or to the possibility of information transfer may be present at birth, over and above recognizing syntax-like structural complexity (ABB). We hope that our results will open the door for future research to approach these intriguing questions.Even though communication is thought to play a role in various models of language evolution16. Taken together, if token variability drove our results, there should have been no difference between the Communicative and the Single Speaker conditions, and if speaker variability were the key factor, there should have been no difference between the Communicative and Echoing conditions. Our findings seem to suggest instead that the newborn brain does not carve out the stimulus space the way a low-level acoustic feature processing account would predict.One possible alternative interpretation of our results could be based on low-level acoustic feature processing, driven by stimulus complexity. Even though, general linguistic complexity was held constant across conditions by the ABB structure, it could be argued that the Communicative condition contained both high speaker variability (like the Echoing condition) and high token variability (like the Single Speaker condition), thus the results reflect the combined effect of these two factors. However, on the basis of our results, such an explanation seems unlikely. Notably, the combined token and speaker variability is identical in the Communicative and the Single Speaker conditions across blocks: not only the number of tokens is the same, but it is either a male or a female voice that can be heard in the Single Speaker blocks. Moreover, it should be assumed that left fronto-temporal areas, which have been identified to track repetitive (ABB) structures and thereby rule-like linguistic complexity, are activated in the Communicative condition over and above the Single Speaker condition exclusively due to the acoustic features of a second speaker. However, acoustic processing takes place in temporal areas, thus if it played a role, we would expect to see it there. In contrast, left inferior frontal areas, including Broca’s area, are sensitive to structural complexity, not to the acoustic features of language29. Two opposing mechanisms may be at play here—however, both of them are different from the above, complexity account. In the Echoing condition the alternation of the female and male voices creates a variable pitch pattern, which may give rise to enhanced prosodic processing at certain locations, while the repetitions may trigger habituation, resulting in decreased activation at other locations. Different areas of the right hemisphere may be more sensitive to one effect or the other, resulting in the opposite pattern of activations. The Communicative condition, by contrast, shares the pitch alternation pattern with the Echoing and the variability of the pseudowords with the Single Speaker condition, so auditorily it does not considerably differ from either, irrespective of the specific mechanism involved.The Echoing condition evoked stronger responses than the Single Speaker condition in right anterior areas, while the opposite pattern was observed in right mid-temporal areas, but crucially, neither control conditions differed from the Communicative condition. These activations are, indeed, likely to reflect auditory and prosodic processes17. The present experiment extends these findings by showing that the same neural systems respond with additional activation when exposed to communication, that is, two speakers producing varying pseudowords in turn-taking alteration with the potential for information transmission. While newborns have been reported to engage in turn-taking vocalizations with their mother30, our result is the first to show their special responsiveness to third-person communicative exchanges. This result demonstrates an extended range of sensitivity to linguistic stimuli when used for communicative purposes, which is likely to provide special learning opportunities both to acquire the structural properties of language and the information content of communication. Our findings open the door for future research to explore the possibility that humans’ dedicated neural mechanisms for language acquisition may be integrated with systems sensitive for ostensive-inferential communication9, which may enable learning the social use of human language rapidly.Previous studies revealed neural systems in the bilateral fronto-temporal areas of the newborn brain that employ dedicated mechanisms to extract linguistic structure from speechThe three conditions Fig. A were prTwenty-one healthy, full-term, monolingual French newborns  participated in this study. Nine additional infants were tested, but not included in the final analysis: four did not finish the experiment due to waking up or becoming fussy, and five were excluded because of technical/stimulus error. Newborns were tested while sleeping Fig. B. Parenthttps://github.com/numediart/MBROLA) both with a female (fr4) and a male (fr3) French voice in a monotonous pitch of 200 Hz and a 150 ms length for all phonemes. In the Communication condition one of the voices uttered one pseudowords token, and the other voice responded with a different token . Question and answer dynamic was achieved by shorter gaps within (400 ms) than between (900–1100 ms) stimulus pairs. In the Echoing condition the first token uttered by the first voice was repeated by the second voice . In the Single Speaker condition the same temporal dynamics were used for each stimulus pair, but only either the male or the female voice spoke within a block . All tokens appeared once in the Communication and the Single Speaker conditions and twice in the Echoing condition (the Echoing condition of each two stimulus lists gives a full token list). Each block consisted of 10 tokens, which yielded 14 blocks per condition and 42 in total. One block lasted about 15 s, which was followed by a 20–22 s break to avoid phase-locking. The experiment as a whole took about 25 min. Blocks, pseudowords, and speaker order were pseudo-randomized using Python 2.7 and the experiment was presented using E-prime 2.10.A 140 tokens of pseudowords of a repetition-based ABB structure  were synthesized using MBROLA  machine  providing 24 channels for measuring hemodynamic responses with a source-detector distance of 3 cm. Pulsating LED sequential illumination (5 mW) in two wavelengths (760 nm and 850 nm) were emitted, and the NIRS signal was recorded at 10.4 Hz sampling rate. Twenty-four channels were established by placing four LED sources and five detectors at analogous locations of the two hemispheres, yielding 12 channels on each side  with dedicated analysis scripts. To reduce the likelihood of false positive results, the permutation tests took into account the autocorrelational properties of the hemodynamic response. The processing steps are described in detail by Abboub et al.13.In order to capture the entire time course of hemodynamic responses to stimulation, analyses were run in the 0–15 s time window after stimulus onset, on both oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb). Low frequency noise  and high frequency noise  were removed by band-pass filtering raw data between 0.01 and 0.7 Hz. To exclude movement artifacts, channels were identified in each block with a concentration change greater than 0.1 mmol × mm over a period of 0.2 s (i.e. two samples) and were rejected. If a channel provided useful data only for less than half of the blocks in a condition, it was discarded. Blocks with excluded channels occurred in a non-systematic  pattern. For each block a baseline level of activity was calculated using a linear fit over the 5 s window preceding the onset and 15 s after the end of the block. Fifteen seconds delay was kept to allow for the hemodynamic responses to settleSupplementary Figure S1.


APC) gene, which encodes a protein necessary for β-catenin degradation, are by far the most prevalent. Other Wnt signaling components are mutated in a smaller proportion of CRCs; these include a FZD-specific ubiquitin E3 ligase known as ring finger protein 43 that removes FZDs from the cell membrane. Our understanding of the genetic and epigenetic landscape of CRC has grown exponentially because of contributions from high-throughput sequencing projects such as The Cancer Genome Atlas. Despite this, no Wnt modulators have been successfully developed for CRC-targeted therapies. In this review, we will focus on the Wnt receptor complex, and speculate on recent discoveries about ring finger protein 43regulating Wnt receptors in CRCs. We then review the current debate on a new APC–Wnt receptor interaction model with therapeutic implications.Hyperactivation of Wnt/β-catenin  signaling in colorectal cancers (CRCs) was identified in the 1990s. Most CRC patients have mutations in genes that encode components of the Wnt pathway. Inactivating mutations in the adenomatous polyposis coli ( Overall survival for CRC patients has steadily increased over the past several decades, mainly because of the success rates of surgical intervention. However, once tumors start to invade local tissues or metastasize to distant sites, 5-year survival rates drop to 71% and 14%, respectively  is the second most common cause of cancer-related death and the third most prevalent malignant tumor worldwide. The American Cancer Society estimated that there would be some 150,000 new CRC cases and about 53,200 CRC-related deaths in the United States in 2020 . In addAPC) gene. Mutations in other Wnt signaling pathway components are also associated with CRCs, although at a lower frequency. Anticancer drugs that target proteins throughout the Wnt signaling cascade are in preclinical and clinical studies, including CRC trials, but no Wnt-targeting therapies are part of current clinical practice. The lack of such therapeutics is partly because of an ongoing debate regarding receptor specificity and general toxicity caused by Wnt inhibition. This review will summarize this debate and discuss recent advances in Wnt receptor research that may inform new targeted therapies for CRC patients.A major therapeutic target in CRC is the frequently mutated and activated Wnt signaling pathway. Hyperactivation of canonical Wnt signaling in CRC is often associated with the mutations inactivating the adenomatous polyposis coli  and Wntless, necessary for the proper secretion of WNT ligands, are located within the endoplasmic reticulum. PORCN first adds lipid modifications to glycosylated WNTs, which are essential for Wntless to bind and transport WNTs to the cell surface and out of WNT-secreting cells  20, 21,,21, 22. Ring finger protein 43 (RNF43) and zing and ring finger 3 (ZNRF3) are direct Wnt targets and negative feedback regulators of Wnt signaling . RNF43 aAPC are by far the most frequent. RNF43 and β-catenin (CTNNB1) mutations are also present in some CRC patients but at a lower frequency. The mutations in either the APC or RNF43 gene cause loss of function. Mutations in CTNNB1 are gain of function: they typically encode forms of β-catenin that are resistant to GSK3-mediated phosphorylation and subsequent ubiquitin-mediated degradation. Mutations in APC, CTNNB1, and RNF43 are virtually mutually exclusive in CRC (A) , c, cAPC archanisms . For exachanisms , 31, 32.APC or CTNNB1 mutations, (B) (C and D), implying that the inhibition of Wnt/β-catenin signaling in CRC patients could help to reduce relapse after treatment. It is also possible that tumor-intrinsic active β-catenin signaling could result in T-cell exclusion and resistance to immunotherapies . H. HAPC orons, (B) , C and Dherapies , 35, 36.Because Wnt signaling regulates cell growth and differentiation, it is not surprising that its dysregulation is commonly associated with tumorigenesis. However, despite substantial efforts, no therapies that target the pathway have been clinically effective. This is not only because of technical challenges and/or the side-effects associated with targeting components of the pathway but also because of the complex regulation of the pathway by many Wnt components. Nonetheless, stabilization of β-catenin, the hallmark of canonical Wnt activation, results in the tumorigenic phenotypes often observed in CRC. So, the stabilized β-catenin remains the most promising target for curing the disease. In the following sections, we will focus on recent discoveries related to RNF43 and APC in CRC and the therapeutic implications of these discoveries.RNF43 frame-shift mutation, G659Vfs  exposure to C59 without showing any phenotypic changes, perhaps because of the expression of drug efflux pumps in Wnt-producing cells  (RNF43 mutations in COREAD samples are not associated with higher Wnt/β-catenin targets (A). In fact, the signet-ring cell carcinoma, a rare subtype of CRC with a high (34.5%) RNF43 mutation rate, actually showed significantly inhibited Wnt/β-catenin signaling than those without RNF43 mutations  of RNF43 could switch it from a Wnt antagonist to a Wnt agonist or may disable RNF43 from suppressing canonical Wnt/β-catenin signaling while leaving its noncanonical Wnt inhibition role intact. However, the noncanonical Wnt activation outcome in this study was limited to DVL2 phosphorylation (RNF43-mutant CRC samples (A)  , 49. Thi targets A. In facutations . One posrylation , and som samples A 37). I. IRNF43 ples (A) . FurtherRNF43 mutations in CRCs and the effectiveness of PORCN inhibitors on a number of RNF43-mediated CRC models provide optimism and opportunity to develop therapeutics targeting Wnt signaling. However, caution is warranted when interpreting the effects of RNF43 mutations, because possibly not all involve loss of function. The impact of RNF43 mutations on noncanonical Wnt signaling also needs to be carefully studied regarding its impact on tumor growth and metastasis. While new efforts are addressing how RNF43 mutations affect Wnt receptor availability and downstream signaling, much work has already been done on how APC mutations regulate CRC initiation and progression. Nonetheless, a novel model involving APC’s interaction with the Wnt receptor complex has been proposed and debated in the field.In summary, the presence of APC is a multifunctional and crucial tumor suppressor gene in the gastrointestinal tract, and mutations causing APC inactivation are found in about 80% of all human colon tumors. Loss-of-function mutations in one allele of APC are widely recognized as the initiating event (“first hit”) in most CRC cases  as well as in CRC animal models. Familial adenomatous polyposis patients with heterozygous germline APC mutations can develop hundreds of colon tumors that are precancerous. Their removal is recommended to avoid progression to invasive and metastatic tumors . Murine Why do mutations in APC result in aggressive colorectal cancer when compared with mutations which directly activate β-catenin? We have long known that human colon cancers having β-catenin mutations grow less aggressively than those with APC mutations . We alsoApc and Apc2 deletion can cause squamous carcinoma with elevated β-catenin levels  is a mammalian APC homolog and closely resembles APC in its overall domain structure. Overexpression of either APC or APC2 in colon cancer cells that lack functional APC could inhibit Wnt signaling effectively , 62. Mamn levels . In agedtivation . Consisty tissue , suggestizations , 67, 68.osophila , whereas-catenin , 70. Alt-catenin , 72 and -catenin , More woIt had been assumed that the loss of APC led to β-catenin stabilization and activation of target genes regardless of upstream receptor status. This assumption was based on the absolute requirement for APC in the destruction complex that targets β-catenin for degradation. However, recent reports have suggested that upstream signals  can still affect Wnt signaling in APC-mutant colorectal and gastric cancer cell lines , 74, 75.et al.  for a met al. .Figure 4APCmin/+ organoids and Drosophila. Importantly, the translational implications from this study included targeting the Wnt receptor complex to inhibit Wnt hyperactivation caused by APC inactivation in cancers like CRC. For example, vantictumab (a pan-FZD antibody), which has been used in clinical trials to treat breast, pancreatic, and ovarian cancers, could be repurposed to treat APC-mutant CRC patients. However, a better strategy is needed to gain more specificity and avoid side-effects caused by a systematic WNT inhibition, such as detrimental effects on bone  or with an LRP6 antibody . This wa on bone . One exa on bone .Developmental Cell  as possible explanations for these differences. Some technical differences between the two studies could also be confounding factors. For example, APC protein truncation by CRISPR could lead to a gain-of-function APC mutant. At the same time, siRNA-mediated APC mRNA loss is probably not as complete as CRISPR-mediated APC truncation. The removal of the entire APC mRNA is not physiologically relevant to human colon cancer patients because most CRC patients retain APC N-terminal fragments. Finally, the levels of APC2 expression in specific cell types (or even subclones of cell lines) may influence the response to loss of APC.i.e., nontumor cells). Additional effort is needed to define further the involvement of endocytosis in Wnt signaling under other contexts.The clathrin-mediated endocytic pathway was proposed to be required for Wnt activation in APC-deficient cells . HoweverIt is crucial to resolve these debates on the requirements for LRP5/6 in APC-deficiency–induced Wnt/β-catenin activation to pursue therapies optimally. The seemingly contradictory mechanisms reported could be because of cell-specific differences in other components of the Wnt pathway (such as APC2 expression level). Further examination of this question could identify a cellular signature that would allow informed predictions of whether specific APC-deficient CRC tumors would be more likely to respond to agents targeting the Wnt receptor complex.Wnt signaling has been intensely studied for several decades. Just when we think that we have a complete understanding of its intricacies, unexpected observations emerge. In addition to the areas outlined above, there are still open questions about the stoichiometry of ligand-receptor complexes. In addition, the broad question of how specificity is generated for downstream signaling by the 19 Wnt ligands, ten Frizzled receptors, and the cohort of additional co-receptors is also of continuing interest. Clarifying these and other questions will undoubtedly identify new opportunities for therapeutic development and keep this research area vibrant for many years to come.The authors declare that they have no conflicts of interest with the contents of this article.


Background Endoscopy training has become increasingly emphasized during general surgery residency as reflected by introduction of the Fundamentals of Endoscopic Surgery (FES) examination, which includes testing of skills on virtual reality (VR) simulators. Although studies exist to assess the ability of the simulator to differentiate between novices and experienced endoscopists, it is not well understood how simulators can differentiate skills among resident cohort.Objective To assess the utility of the VR simulator, we evaluated the correlation between resident endoscopy experience and performance on two VR simulator colonoscopy modules on the GI-BRONCH Mentor .Methods Postgraduate years 2 to 5 residents completed “easy” and “difficult” VR colonoscopies, and performance metrics were recorded from October 2017 to February 2018 at Rutgers' two general surgery residency programs. Resident endoscopy experience was obtained through Accreditation Council for Graduate Medical Education case logs. Correlations between resident endoscopy experience and VR colonoscopy performance metrics were assessed using Spearman's rho (ρ) correlation statistic and bivariate logistic regression.Results Fifty-five residents out of 65 (84.6%) eligible participants completed the study. There were limited correlations found between resident endoscopy experience and FES performance metrics and no correlations were found between resident endoscopy experience and binary metrics of colonoscopy—ability to complete colonoscopy, ability to retroflex, and withdrawal time of less than 6 minutes.Conclusion The VR simulator may have a limited ability to discriminate between experience levels among resident cohort. Future studies are needed to further understand how well the VR simulator metrics correlate with resident endoscopy experience. Over the past several years, there has been an increased emphasis placed on the importance of endoscopy during general surgery training. This has been reflected by the Accreditation Council for Graduate Medical Education (ACGME) increasing the number of endoscopy cases required for general surgery trainees as well as the introduction of the American Board of Surgery (ABS) Flexible Endoscopy Curriculum, which culminates in the Fundamentals of Endoscopic Surgery (FES) examination. The ACGME and the ABS utilize resident operative experience as a proxy for procedural competence. As of 2018, the above-mentioned requirements must be met to sit for the general surgery boards.The FES examination consists of a multiple-choice cognitive examination and hands-on skills test.As the FES skills examination is administered on the GI Mentor, there is a theoretical advantage of using this platform to prepare for this examination; it could be used to allow a resident to gauge their readiness to take the examination and practice their skills. One downside is that the specific skills modules tested on the FES examination are not publicly available.To assess the utility of the VR platform, we aimed to evaluate the correlation between resident clinical endoscopy experience and performance on two selected VR colonoscopy modules at two general surgery residency programs. We hypothesized that performance on a simulated colonoscopy would be a poor way to discriminate between experience levels among resident cohort.The resident endoscopy curriculum includes review of the FES online didactic curriculum during the postgraduate years (PGYs) 1 through 5, which was historically complimented by dedicated clinical endoscopy experience from PGY 2 through PGY 5. When the GI Mentor became available on the two Rutgers campuses, New Jersey Medical School (NJMS) and Robert Wood Johnson (RWJ), it was incorporated into the formal simulation curriculum at each general surgery residency training program. After this implementation, these data for this study were collected. Study participation was voluntary, and trainees were offered the same orientation to the simulator regardless of participation. The study took place from October 2017 through February 2018. PGY 2 through PGY 5 categorical residents at NJMS and RWJ general surgery residency programs were eligible for participation. The only exclusion criteria was performing deliberate practice on the simulator prior to study enrollment out of concern that the participants might thoroughly know the simulations, which would be an inaccurate reflection of endoscopic skill. Deliberate practice was identified as those residents who had either already taken the FES examination or used the same modules—the practice modules and/or case modules—to prepare for the examination.not comfortable) to 5 (very comfortable) with performing upper and lower endoscopies, and remark on their prior experience with endoscopy simulators. Upper endoscopy experience was asked to participants as there are data that show a possible correlation between upper and lower endoscopy performances.Participants were asked to complete a survey to provide demographic information, rate their comfort level on a scale of 1  and “difficult”  virtual colonoscopies. They: (1) took a picture of the ileocecal valve, (2) performed a retroflexion maneuver at the end of the procedure and took a picture for the “easy” VR colonoscopy, and (3) did not intubate the terminal ileum. Proctors gave participants 15 minutes to complete each colonoscopy. This 15-minute time limit was chosen to allow for ample time for even novices to complete both cases while considering the time constraints unique to general surgery. Proctors were general surgery residents in a research year and were well oriented to the details of the selected study cases.Participants were tasked with completing the “easy” , percentage of mucosal surface examined (%), time to reach the cecum (seconds), and total time (seconds). The amount of additional time that was spent performing the retroflexion maneuver was included by the simulator in the “total time” metric. Therefore, the metrics dependent on “total time”—calculated withdrawal time and efficiency—would not be accurate and were excluded. Each proctor recorded whether the colonoscopy was completed. Metrics assessed for the “difficult” VR colonoscopy included those of the “easy” VR colonoscopy along with efficiency of screening and withdrawal time. Withdrawal time (seconds) was calculated using the recorded time to cecum and total time metrics. For other binary metrics, the ability to successfully retroflex was assessed for the “easy” VR colonoscopy and having a withdrawal time > 6 minutes—which is commonly cited as a quality indicator for colonoscopyParticipants had their deidentified ACGME case logs downloaded from the secure ACGME Case Log System by the residency program coordinators at each site. All upper and lower endoscopy cases performed up until the date of study participation were counted. Upper endoscopy cases included all diagnostic and/or therapeutic endoscopies of the esophagus, stomach, and/or small bowel. Lower endoscopy cases included all diagnostic and/or therapeutic sigmoidoscopies or colonoscopies.p-value of < 0.05 was considered significant.Descriptive statistics for all variables are reported as counts with percentages for categorical variables and as medians with interquartile ranges for continuous variables. Correlations between clinical endoscopy experience—upper, lower, and total endoscopy cases—and continuous performance metrics were assessed using Spearman's rho (ρ) correlation statistic. The correlations between clinical endoscopy experience and binary performance metrics—the ability to complete a given colonoscopy, retroflex in the “easy” VR colonoscopy, and have a withdrawal time > 6 minutes in the “difficult” VR colonoscopy—were assessed using bivariate logistic regression.SAS software Version 9.4  was used for statistical analysis.Approval from the Institutional Review Board was obtained from both Rutgers sites—RWJ and NJMS.Fifty-five out of 65 possible residents (84.6%) met criteria and completed the study . While p = 0.04). Prior physical simulator use  and right handedness were correlated with a longer withdrawal time.For the “easy” VR colonoscopy, the only significant correlation found was between number of upper endoscopies performed and percentage of mucosa evaluated . HoweveAs one of the largest studies that looked at the correlation of documented resident endoscopy experience with performance on a simulator, our study showed limited correlations between upper, lower, and total endoscopy experience and metrics. Overall, we did not find any correlation with endoscopy experience and the main metrics—completing the colonoscopy, the ability to successfully retroflex, withdrawal time—and most of the FES examination skills. We found that for the “easy” VR colonoscopy, there was a correlation between one's upper endoscopy experience and percentage of mucosa evaluated; several studies have shown a correlation between total endoscopy numbers and this particular metric,Given the correlation between endoscopy experience—upper, lower, and total—and faster time to cecum with the more “difficult” VR colonoscopy and the fact that the critical difference between the two cases was that the “difficult” one was prone to loop formation, this suggests that those with more clinical endoscopy experience are better able to recognize loop formation and/or reduce loops, thus resulting in a faster time to cecum. The metric of efficiency is a combination of time to cecum and percentage of mucosa evaluated; since there was no correlation with endoscopy experience and percentage of mucosa evaluated, this suggests that the correlation between endoscopy experience and efficiency is heavily influenced by the correlation between endoscopy experience and time to cecum. Prior physical model use was correlated with a longer withdrawal time. The reason for this may be an increased familiarity with the expectation to inspect the mucosa carefully for lesions during withdrawal. Although right handedness was correlated with a longer withdrawal time, this finding should be interpreted with caution as there were 51 right-handed versus only 4 left-handed residents.Analysis of our dataset reveals that the number of endoscopy cases completed by our residents showed a right skew, with the majority falling into the 0 to 9 cases category. Part of the reason for this was the reduced number of PGY 5. However, this may be a more realistic distribution of trainee experience. Studies using the GI Mentor test modules showed an ability to distinguish between novices and experienced endoscopists.There were limitations in our study. For one, our study primarily focuses on a limited number of components of validity evidence. According to the most current edition of the American Educational Research Association and American Psychological Association Standards for Educational and Psychological Testing, validity evidence can be broken down into five components: content, response processes, internal structure, relations with other variables, and consequences.Another limitation of our study is that the ACGME case logging system has been shown to be limited by some degree of inaccuracy secondary to underreporting and inaccurate logging of procedures by trainees.A future step would be to further analyze virtual colonoscopy performance within a resident cohort for any potential significant differences with regard to targeting ability—the fifth FES skill that can now be assessed on the simulator.In conclusion, we did not find correlations between resident endoscopy experience and the main metrics of colonoscopy—completion of colonoscopy, the ability to successfully retroflex, and withdrawal time. There were also limited correlations found with resident endoscopy experience and continuous metrics. As the FES examination is used for general surgery residency programs throughout the country, it is imperative to understand the real utility of simulators for surgical training evaluation.


To know its hitherto unidentified microbiome profile, we employed high-throughput sequencing to uncover the bacteriome and mycobiome in fresh and 12-h fermented samples. Fresh Kalparasa had a pH of 7.2, which dropped to 4.5 after 12 h, signifying fermentation of the sap. Diversity analysis indicated fresh Kalparasa having higher bacterial species than the fermented one. Contrary to this, fresh sap had lower fungal/yeast diversity than the fermented sample. Fresh Kalparasa had relatively higher abundance of probiotic-type Leuconostoc genus followed by equal proportions of Gluconobacter, Acetobacter, and Fructobacillus. The 12-h fermented Kalparasa showed a significant increase in Gluconobacter with a sharp decrease in Leuconostoc. Mycobiome data revealed fresh Kalparasa to be preponderant in Saccharomyces and Hanseniaspora genera of yeasts while the fermented sap had higher representation of Hanseniaspora and Cortinarius and lesser Saccharomyces. This suggested that the fermentation of Kalparasa was probably driven by symbiotic culture of bacteria and yeasts (SCOBY), particularly acetic acid bacteria and non-Saccharomyces yeasts. The bacteriome-function predictions highlighted the enrichment of glycerophospholipid, ABC transporters, purine, and pyrimidine metabolisms. Based on our findings, Kalparasa containing large population of Leuconostoc mesenteroides, Fructobacillus fructosus, Saccharomyces cerevisiae, and Hanseniaspora guilliermondii can be promoted as a healthy “unfermented” plant edible food containing live probiotic-type microbiome during its consumption.The phloem sap tapped from unopened inflorescence (spadix) of coconut palm using a novel collecting device, “coco-sap chiller,” has been branded Kalparasa Borassus flabellifer) (Cocos nucifera L.) (Elaeis guineensis) (Xylem and phloem saps are watery fluids  having aellifer) , coconutfera L.) , Africanneensis) , and datneera) is generally collected in open mud pots or plastic containers. The collection methods and collecting units such as open earthen pots traditionally used , and vinegar.Non-availability of proper collection apparatus and fermentation inhibitors resulting in quick conversion to alcoholic beverage of the coconut inflorescence sap made its tapping an offense in many of the coconut-growing states in southern India as per the Excise Acts . The palneera collection device, coco-sap chiller (under Indian patent consideration) by ICAR-Central Plantation Crops Research Institute , literally meaning “juice or flavor of the Kalpavriksha—the coconut palm.” Significant differences in the physical, chemical, biochemical, and microbiological properties of Kalparasa®  and traditionally collected neera in earthen pot were recently reported  isolated from the naturally fermenting coconut neera were reported  studies could highlight the superior biochemical properties of Kalparasa in terms of sugars, amino acids, vitamins, phenols, and flavonoids  was regularly done. The pH of the soil was 5.6 with 0.6% OC content. As the palms were not affected by any serious pests or diseases, no major plant protection measures were taken for at least 3 years prior to tapping of the palms other than preventive cares.Kalparasa for metagenomic analysis was initiated during the November to December period as per the standardized protocol described using the coco-sap chiller  were strategically placed on suitable canopy parts (mainly fronds) for about 15 min to allow deposition of any aerially moving microbiota that could enter the coco-sap chiller while fixing it to spadix. At least two plates for each agar medium were placed. The plates were closed with lids after the exposure period, placed into sterile polythene bags, tied and sent down to ground, and were immediately transferred to the microbiology laboratory for the incubation at 28°C ± 2°C for 48–72 h for scoring the growth of bacterial and fungal colonies and 5 days for actinomycetes colonies.Kalparasa harvested overnight was removed from the coco-sap chiller, brought down from the palm, and right away transferred to the microbiology laboratory. The pH and color of the samples were immediately checked to confirm the quality of the Kalparasa. A sensory evaluation was also done to assure that the sap was fresh, aromatic, and unfermented. About 50 ml of the sub-sample from each bag was simultaneously taken out for culture-dependent microbial analysis. About 50 ml (×2) of the samples was transferred to sterile centrifuge tubes (50 ml) and kept at –45°C for the culture-independent metagenome study. Another 250 ml of the sub-sample was kept in a sterile plastic bottle for overnight fermentation (12 h). Thus, we had two different treatments to analyze: fresh and 12-h fermented Kalparasa. The next morning, 50 ml of sub-sample from each of the three bottles was immediately subjected to culture-dependent microbial analysis and 50 ml was transferred to sterile centrifuge tubes and stored at –45°C for the metagenomic study.The polythene bag containing the Kalparasa from freshly collected and 12-h fermented samples were serially diluted and pour plated in different agar media to enumerate the bacterial, fungal, yeast, and actinomycetes populations. The plates were incubated for different time periods and then observed for colony-forming units of the microbial communities.For culture-dependent analysis, 10-ml aliquots of Kalparasa samples was used for total DNA extraction using the DNeasy Power Food Microbial kit (Qiagen) following the manufacturer’s protocol. The extracted DNA was quantified using Nanodrop  and Qubit  and further processed immediately for sequencing. Paired end (250 × 2) 16S rRNA amplicon sequencing of the V3–V4 region was performed for the bacterial diversity analysis of the Kalparasa. The primers employed for amplifying the V3–V4 variable region were 341F: CCTACGGGNGGCWGCAG and 805R: GACTACHVGGGTATCTAATCC; for the ITS: ITS1F: CTTGGTCATTTAGAGGAAGTAA and ITS2R: GCTGCGTTCTTCATCGATGC. To begin, for having sufficient concentrations of the target fragments, repeated amplification for each sample was done with the following PCR conditions: pre-amplification denaturation at 95°C for 3 min; 25 cycles (35 cycles for ITS) of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and extension at 72°C for 10 min followed by holding at 4°C. The amplicons were then pooled in equimolar concentrations, cleaned up, purified using Agencourt AM pure magnetic beads , and then quantified using Qubit. The pooled PCR amplicon samples were sequenced on Illumina HiSeq following the sequencing procedure of For metagenomic analysis, 50 ml of fresh and 12-h fermented The raw 16S rRNA and ITS amplicon sequencing data for bacteria and fungi, respectively, were processed using the DADA2 v1.10.1 analysis pipeline package for modeling and correcting Illumina-sequenced amplicon errors  in R v 3The de-multiplexed HQ processed data were further handled using the Bioconductor package phyloseq v1.26.0 . In ordeIn order to predict the functional metagenomic content of the bacterial community over the samples, the ASVs were submitted to the Piphillin server , which iAll statistical computations were performed using R packages . BacteriKalparasa samples, three fresh  and three fermented for 12 h , were analyzed for microbial properties using culture-dependent and -independent methods. Before their analysis, the microbial profile of the aerial environment of the coconut canopy was determined using a culture-dependent procedure. The coconut canopy aerial microbiota on the day of fixing the coco-sap chiller was found to be sparse for all the three palms  selected for Kalparasa tapping. Exposure of agar plates to catch the airborne microbiota in the coconut canopy resulted in the growth of one bacterial, one fungal, and three yeast colonies in the respective media (data not furnished). On the other hand, microbial status of the freshly collected and 12-h fermented Kalparasa indicated the presence of bacteria  and yeasts in both samples. The population of yeasts was significantly higher in the fermented samples (CS-12H: 15×107 cfu ml–1) compared to fresh Kalparasa (CS-F: 6×104 cfu ml–1). The pH of the fresh Kalparasa was 7.2, and it dropped to 4.5 after 12 h of keeping in ambient conditions.A total of six Kalparasa, the V3–V4 variable region of 16S rRNA was amplified for all the six samples. After pre-processing the raw data, a total of 1,192,109 chimera-free reads were obtained  had highest abundance of the genus Leuconostoc (61.15%) followed by Acetobacter (10.8%), Gluconobacter (10.49%), and Fructobacillus (7.74%). The 12-h fermented Kalparasa (CS-12H) had a much lower Leuconostoc content (39.3%) and lower Acetobacter (7.20%) but three times more Gluconobacter (31.52) and twice higher Fructobacillus (15.47%) compared to fresh Kalparasa , orders (11), families (25), genera (31), and species (40) were much higher than bacteria  and Lachancea (7.1%) and less of Hanseniaspora (28.4%), Cortinarius (7.55%), and Candida (1.22%). CS-12H had double the abundance of Hanseniaspora (42.63%), higher Cortinarius (22.1%) and Candida (17.82), and very low abundance of Saccharomyces (12.04) and Lachancea (2.59%)  and fermented samples (CS-12H3). A total of 1,771,246 chimera-free reads were generated with CS-12H having higher numbers  than CS-F  . The Venbacteria . The mos (2.59%) . Both fralparasa . Log2-foalparasa . The genmeasured  with themeasured . The divmeasured . We gene dataset .Kalparasa had a very abundant bacterial activity directed toward the pathways , coconut palm (C. nucifera), raffia palm (Raphia hookeri), oil palm (Elaeis guineensis), and date palm (Phoenix dactylifera), is tapped from their unopened inflorescences in several regions in South Asia, Southeast Asia, the Caribbean, South and North America, Africa, and Micronesia for consumption as locally produced alcoholic beverages. The sap is also a rich source of nutrients, minerals, and secondary metabolites, which makes it an edible plant juice. In India, the age-old practice of non-destructive tapping of the inflorescence was adopted to release the phloem sap of coconut and palmyra. The tappers tied an earthen pot, smeared on the inside with lime, to the inflorescence for collecting the sweet sap. The lime helps in slowing down fermentation of the sap. However, despite its application, fermentation begins the moment the sap starts trickling out from the inflorescence into the pot . The nutritional quality in terms of amino acid, minerals, vitamins, phenols, and secondary metabolites along with the basic microbial profile of Kalparasa was studied in detail, which bolstered its distinctiveness and health-promoting properties , represented more than 60% by the Leuconostoc genus, with L. mesenteroides being the prominent species. Majority of the ASVs were common to both fresh and fermented Kalparasa, while 71 were observed in fresh and 44 were observed in fermented sap. This was indicative of the minimum diversity difference in both the samples and the only difference was the enrichment of species as the fermentation progressed. In a report involving the study of biochemical and microbiological properties of naturally fermenting sap tapped from three different palms , it was observed that Fructobacillus and Leuconostoc spp. were preponderant during the 30 days of experiment. The freshly collected sap was acidic in nature with pH ranging between 3.74 and 3.84   and Proteobacteria (44.06%) was closer to each other in the fermented sample. The abundance of the bacterial genus Gluconobacter was the highest followed by Fructobacillus, whereas Leuconostoc was reduced to almost half compared to fresh Kalparasa. As the fermentation of Kalparasa proceeded, microbiota dynamics showed partial replacement of Leuconostoc, Acetobacter, and Acinetobacter groups with Fructobacillus, Gluconobacter, and Lactococcus. In the case of tuba, with increase in the fermentation period, the abundance of Fructobacillus spp. increased. So, a clear difference in the bacterial profile in Kalparasa extracted from coconut palms using the coco sap chiller and that of tuba harvested by the traditional method in Mexico was observed. However, our observations of high sucrose content and Leuconostoc genus in fresh Kalparasa matched with that of aguamiel, which is fresh sap collected from agave  had shown the presence of seven different bacterial species including Lactobacillus, Leuconostoc, and Enterococcus spp. displaying very good probiotic characteristics such as antibiotic traits and hydroxyl radical-scavenging attributes, among others , like Leuconostoc, had also been isolated from the nectar of Butea monosperma flower already reported for its probiotic potential  . Leuconoom agave . Both Legetables . Not onl tequila . In palmteroides  that coutabolite . It is, g others . They we ability . Kimchi, studies . The souentation . In addiotential . Fructopotential . These pKalparasa had more diverse constituents compared to the bacteriome. The mycobiome was represented by 31 genera and 40 species of fungi, whereas the bacteriome was made up of 16 unique genera and 18 species. Saccharomyces, Hanseniaspora, and Lachancea were the important yeast genera present in the Kalparasa at both stages of analysis, albeit with differences in their population. The dominant species were S. cerevisiae, H. guilliermondii, Lachancea fermentati, C. saturatus, and C. tropicalis. Fresh Kalparasa had higher abundance of Saccharomyces and Lachancea compared with the fermented sample, which had double the abundance of Hanseniaspora, Candida, and Cortinarius and three-fourths less of Saccharomyces compared with the fresh sample. There appeared to be a high number of unique ASVs in the fermented sap than the fresh Kalparasa as indicated by the Venn diagram. Population of yeasts was always found in higher abundance in fermented foods, even in coconut sap  in Mexico, it was observed that H. guilliermondii and C. tropicalis were the main yeasts in all the three palm trees sampled whereas S. cerevisiae was found only in two of them  as observed in several beverages  and equal proportions of genetic information processing, environmental information processing, and cellular processes. This was obvious because the Kalparasa was rich in sugars, amino acids, and phenols  was also seen as a dominant metabolic activity in both the samples of the Kalparasa. Yeasts have been reported to be involved in QS in fermented food for possibly regulating their community structure  normally associated with the traditional method of sap collection. A better understanding of the microbiome can be obtained by analyzing Kalparasa from more number of coconut palms along with the associated plant parts.Thus, metagenomic analysis conducted by us with limited palm samples indicated the presence of abundant and diverse microbiome in fresh unfermented Kalparasa, it could be worthwhile to examine its effect on cell longevity.Longevity of any cell or its lifespan is determined by the period over which the constituent cells remain metabolically functional. Palms, such as coconut, have primary growth living cells that remain metabolically active throughout its lifetime. In a broad sense, palm cells, particularly the sieve tubes and their companion cells, are therefore deemed immortal . Though Kalparasa disclosed the elaborate diversity of bacteria and yeasts harbored in the coconut inflorescence sap collected using the novel “coco-sap chiller.” Fresh Kalparasa had a pH of 7.2, indicating the non-fermented status, accompanied with high abundance of lactic acid bacteria (LAB), Leuconostoc spp., and Fructobacillus spp., and yeasts such as Saccharomyces spp., Hanseniaspora spp., and Lachancea spp. The dominant species were L. mesenteroides, F. fructosus, S. cerevisiae, and H. guilliermondii in fresh Kalparasa. Keeping the Kalparasa in ambient conditions for 12 h resulted in lowering of pH to 4.7 accompanied with sour alcoholic aroma indicating fermentation. A shift from LAB to acetic acid bacteria (AAB) with Gluconobacter and Fructobacillus spp. dominating the bacteriome density while Hanseniaspora spp. was highest in mycobiome demonstrated that AAB and non-Saccharomyces yeast could be driving the fermentation of Kalparasa. This reflected that the fermentation could be driven by SCOBY. Our studies clearly indicated that like fermented palm saps harbored probiotic microbiome, fresh Kalparasa also had abundant probiotic-type microbiome consisting of L. mesenteroides, F. fructosus, S. cerevisiae, and H. guilliermondii. These findings add value to promote Kalparasa as a natural health drink full of beneficial properties arising from its probiotic-type microbiome.The 16S rRNA and ITS amplicon sequencing-based metagenomic analysis of PRJNA701871.The datasets presented in this study can be found in NCBI BioProject ID: Kalparasa, and MA were involved in the investigation. MG, SS, and AG wrote the manuscript with inputs from all authors. All the authors contributed to the article and approved the submitted version.MG and AG designed and conducted the study and obtained the data. SS and MG carried out the bioinformatic analysis. SS did the statistical analysis in R-package. KBH, the original developer of coco-sap chiller and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Channa argus) is an economically important freshwater fish native to East Asia. To explore potential interaction between snakehead MC4R and MRAP2, herein we cloned snakehead mc4r and mrap2. The snakehead mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids, while snakehead mrap2 contained a 693 bp open reading frame encoding a protein of 230 amino acids. Synteny analysis indicated that mc4r was highly conserved with similar gene arrangement, while mrap2 contained two isoforms in teleost with different gene orders. Snakehead mc4r was primarily expressed in the brain, whereas mrap2 was expressed in the brain and intestine. Snakehead mc4r and mrap2 expression was modulated by fasting and refeeding. Further pharmacological experiments showed that the cloned snakehead MC4R was functional, capable of binding to peptide agonists and increasing intracellular cAMP production in a dose-dependent manner. Snakehead MC4R exhibited high constitutive activity. MRAP2 significantly decreased basal and agonist-stimulated cAMP signaling. These findings suggest that snakehead MC4R might be involved in energy balance regulation by interacting with MRAP2. Further studies are needed to elucidate MC4R in regulating diverse physiological processes in snakehead.The melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of MC4R. The Northern snakehead ( MC4R and common genetic variation near MC4R lead to a dominant form of obesity and insulin resistance -NDP-MSH and [125I]-cAMP were iodinated in our lab using chloramine T method [NDP-MSH was purchased from Peptides International , α- and β-MSHs were purchased from Pi Proteomics , and ACTH (1–24) was purchased from Phoenix Pharmaceuticals . Both [T method ,43. The T method . The codmc4r, mrap2a, and mrap2b as references. After searching, we obtained the genomic and transcriptomic sequences of the snakehead mc4r and mrap2  genes. Two pairs of primers were designed to validate the two sequences, respectively. Total RNA was isolated from the whole brain with the Trizol reagent  according to manufacturer’s protocol, and 1 μg of the RNA was reverse transcribed to cDNA using Super ScriptTM II RT reverse transcriptase . The basic cycling conditions of the PCR were set as follows: A denaturing stage at 94 °C for 30 s, gene-specific annealing temperature for 45 s and an elongation stage at 72 °C for 60 s, a total of 34 cycles. The PCR products were purified from agarose gel using the Universal DNA Purification Kit , and then cloned into the pMD-19T vector (TaKaRa). The inserts were sequenced at BGI , and the primers used to validate mc4r and mrap2 genes were listed in Protein similarity-based blast was performed to search the target genes in the snakehead genome and transcriptome databases , using zhttps://www.ncbi.nlm.nih.gov/gorf/gorf.html), and then the putative protein sequences were translated with Primer Premier 5.0 software. Moreover, the Bioedit software was used to determine the isoelectric point (PI), the online tool TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) was used to identify the transmembrane domain, and the Expasy (http://www.expasy.org/tools/) was used to predict glycosylation and phosphorylation sites. Furthermore, multiple sequence alignments were done as described in our previous work [https://www.swissmodel.expasy.org/) using human MC4R as reference [MC4R and MRAP2 in vertebrates as described previously [The coding sequences (CDS) were determined using online software ORF finder .Extraction of total RNA from fish tissues and first strand cDNA synthesis were performed as described above. Then, real-time PCR was used to detect the mRNA expressions of eviously , and tub2-humidified atmosphere. The cells were plated into 6-well or 24-well plates  pre-coated with 0.1% gelatin and transfected or co-transfected when cells reached at ~70% confluence using calcium phosphate precipitation method [Human Embryonic Kidney (HEK) 293T cells, purchased from American Type Culture Collection , were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen) containing 10% newborn calf serum , 10 mM HEPES, 0.25 μg/mL of amphotericin B, 50 μg/mL of gentamicin, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% COn method .To investigate the effect of snakehead MRAP2 on the cell surface and total expression levels of MC4R, cells were plated into 6-well plates and transfected with snakehead MC4R and MRAP2 plasmids in four ratios . Then, cells were incubated with mouse anti-myc 9E10 monoclonal antibody  diluted 1:40 and Alexa Fluor 488-labeled goat anti-mouse antibody (Invitrogen) diluted 1:2000 for 1 h, respectively. Flow cytometry was carried out, and C6 Accuri Cytometer  was applied to collect fluorescence signals. The expression level of caMC4R (caMC4R fluorescence − pcDNA3.1 fluorescence) from 1:0 (caMC4R/caMRAP2) group was set as 100%, and the expression levels of other groups were calculated as a percentage of 1:0 group .To assess the ligand binding properties of the caMC4R, four ligands , α-MSH, β-MSH) were used. Forty-eight hours after transfection, HEK293T cells were washed twice with warm DMEM containing 1 mg/mL bovine serum albumin  (DMEM/BSA). To study the effects of caMRAP2 on the binding property of caMC4R, two ligands (α-MSH and ACTH (1–24)) were used. Forty-eight hours after transfection, HEK293T cells co-transfected with caMC4R and caMRAP2 at four ratios  were washed twice with warm DMEM/BSA. After 1-h incubation with ligands, the competitive binding reaction was terminated by washing cells twice with cold Hank’s balanced salt solution containing 1 mg/mL BSA on ice. The cells were lysed with 0.5 M NaOH, collected with cotton swabs, and radioactivity detected with gamma counter .−12 to 10−6 M for NDP-MSH and ACTH (1–24) or from 10−11 to 10−5 M for α-MSH and β-MSH. After an hour’s incubation at 37 °C, the reaction was terminated on ice and the intracellular cAMP was collected by adding 0.5 M perchloric acid containing 180 μg/mL theophylline (Sigma–Aldrich) and 0.72 M KOH/0.6 M KHCO3 into each well. The cAMP levels were measured by radioimmunoassay (RIA) [Forty-eight hours after transfection, HEK293T cells were washed twice with warm DMEM/BSA and then incubated with DMEM/BSA containing 0.5 mM isobutylmethylxanthine  at 37 °C for another 15 min. Then, different concentrations of NDP-MSH, ACTH (1–24), α-MSH, or β-MSH were added to make the final concentration of ligands ranging from 10ay (RIA) .max) of cAMP levels to α-MSH and ACTH (1–24) stimulation, cells were co-transfected with caMC4R and caMRAP2 in four ratios . To study the constitutive activity of Gs cAMP, cells were transfected with different concentrations caMC4R plasmid .To investigate the potential effect of caMRAP2 on caMC4R signaling, two ratios of caMC4R and caMRAP2 (1:0 and 1:5) were applied to co-transfect cells, and two ligands, α-MSH and ACTH (1–24), were used. To explore the dose-dependent effect of caMRAP2 on the maximal response , followed by the post hoc test (least significant difference test and Duncan’s multiple range test), after confirming for data normality and homogeneity of variances. GraphPad Prism 6.0 software  was used to calculate the parameters of ligand binding and cAMP signaling assays including Bmc4r was 1515 bp in length, and contained a 984 bp open reading frame (ORF) encoding a putative 327-amino acid protein, and has been deposited into the GenBank database (accession number KU728167) (The cDNA sequence of snakehead U728167) A.mc4r, the coding region of the snakehead mc4r was intronless. Potential glycosylation and phosphorylation sites were found in N- and C-termini, respectively  and a living fossil species reedfish . However, mrap2a was lost in Perciform fishes including snakehead (C. argus), tilapia (O. niloticus), and pufferfish (T. rubripes). Interestingly, a different gene cluster SOBPb-MRAP2b-RIPPLY2-SIM1b was identified in almost all teleosts examined in the present study, except reedfish  method based on a series of protein sequences of vertebrate MC4Rs and MRAP2s, and both trees were robust with high bootstrap values .Dicentrarchus labrax) and large yellow croaker (Larimichthys crocea) MC4Rs (As shown in a) MC4Rs A. Additia) MC4Rs B. Notabla) MC4Rs B.mc4r and mrap2 was determined using quantitative real-time PCR. Similar to other fish, snakehead mc4r was found to be extensively expressed in central and peripheral tissues, including adipose, brain, eye, gill, gonad (ovary), heart, intestine, kidney, liver, muscle, and spleen  , α-MSH and β-MSH) were used to compete with f hMC4R) .125I-NDP-MSH, caMC4R showed significantly decreased or increased IC50 values in comparison with hMC4R, whereas when the other two ligands were used as competitors, hMC4R and caMC4R exhibited similar IC50s (When NDP-MSH or α-MSH was used to displace the ar IC50s .50 of ACTH (1–24) for caMC4R was significantly lower than that of hMC4R, whereas the EC50s of NDP-MSH, α-MSH, and β-MSH were comparable with those of hMC4R  was decreased by caMRAP2 in the 1:3 and 1:5 groups, whereas no change was observed in IC50s for α-MSH among the four groups  with other teleost MRAP2s, suggesting MC4R was more conserved than MRAP2 in teleosts.In this study, we cloned snakehead mc4r was identified in vertebrates with a conservative gene cluster RNF152-CDH20-MC4R-DROSHA-CDH6, suggesting that this receptor is highly conserved and may play similar roles in vertebrates. In tetrapod, only a single copy of mrap2 was identified in their genomes, exhibiting a conserved gene cluster NT5E-TBX18-CEP162-MRAP2-CYB5R4-RIPPLY2. In teleosts, the gene number of mrap2 is variable. In zebrafish, two copies of mrap2 (mrap2a and mrap2b) were identified, and functional studies showed that MRAP2a decreases binding affinity for α-MSH while MRAP2b increases ligand sensitivity of MC4R [mrap2 was identified and it shares a similar conserved genetic locus with tetrapod mrap2 and zebrafish mrap2a, suggesting that the duplication of mrap2 might have occurred after the specific whole genome duplication event in teleost [mrap2a was lost in Perciform and Tetraodontiform fishes, including snakehead, tilapia, and puffer fish. However, the mrap2b paralog was retained in these teleost genomes with a conserved gene cluster SOBPb-MRAP2b-RIPPLY2-SIM1b, suggesting the evolutionary process of mrap2 is complex in teleosts and the physiological role of this gene might be variable.Comparative gene synteny analysis showed that only a single copy of  of MC4R . In reed teleost . InteresTo investigate the evolutionary relationship of vertebrate MC4Rs and MRAP2s, phylogenetic trees were constructed. Both trees were divided into two subgroups of teleost and tetrapod subgroup, similar to previous studies ,32,33. Fmc4r was widely expressed in various tissues with high expression in brain and gonad, similar to previous studies [mrap2 was also extensively distributed in several tissues, with relatively high expression in brain and gonad. This distribution pattern of mrap2 was also found in orange-spotted grouper [mc4r and mrap2 exhibited similar distribution pattern, indicating that they might interact with each other in snakehead.Data from real-time quantitative PCR showed that  studies ,32,33,56 studies ,32 and s studies ,57. Mean grouper , Nile ti grouper , and top grouper , suggestmc4r and mrap2 expression after short-term and long-term fasting and refeeding. We showed that expression of these genes was modulated by these manipulations  had 3-fold higher potency at caMC4R than hMC4R. Previous studies demonstrated that fish MC4Rs have decreased binding capacities [To investigate the pharmacological characteristics of caMC4R, four ligands were used, together with hMC4R for comparison. Our results demonstrated that caMC4R was fully functional. The synthetic agonist NDP-MSH bound to caMC4R with the highest affinity with an ICpacities ,30,31,60A remarkably elevated basal cAMP activity was found in caMC4R. This phenomenon has also been reported in other fish species, such as spotted scat , grass c50 of caMC4R to α-MSH. In topmouth culter, two isoforms of MRAP2 are also shown to increase binding affinity of MC4R to ACTH (1–24), but not to α-MSH [50 of caMC4R in response to α-MSH or ACTH (1–24) was observed. Previous studies demonstrated that MC4R can become an ACTH receptor by increasing ligand sensitivity to ACTH in the presence of MRAP2 [In this study, we showed that caMRAP2 did not affect cell surface and total expression of caMC4R, similar to the observation in chicken , but difto α-MSH . In addito α-MSH . MRAP2 mto α-MSH  and topmto α-MSH . In the of MRAP2 ,59,65. Imc4r copies in larger males act as dominant-negative mutants and delay puberty onset [gnrh expression in the hypothalamus, thereby modulating pituitary FSH and LH synthesis [gnih and decrease sgnrh and cgnrh expression in vitro; they also affect expression of some ovarian genes. These studies demonstrated that MC4R could play a vital role in reproductive regulation in fish. In the present study, we also observed that mc4r and mrap2 were highly expressed in gonad, implying a potential role in reproduction. Therefore, future studies on MC4R regulation of reproductive function are warranted.In addition to energy homeostasis, MC4R also regulates reproduction and sexual function . Comparety onset . In spotynthesis . In blacIn conclusion, our studies on snakehead MC4R and MRAP2 and their interaction contributes to a better understanding of fish MC4Rs. Some aspects, such as high expression of these genes in the brain and significant expression in the gonad, and high basal activity were similar to previous studies in fish. However, our data, combined with literature, suggested that functional implications of MRAP2 on MC4R are divergent in different species. Whether MC4R could be modulated to improve growth and reproduction of snakehead remains to be investigated.